Cd147 as receptor for pilus-mediated adhesion of meningococci to vascular endothelia

ABSTRACT

The present invention concerns the use of an inhibitor of an interaction between between type IV pilus-associated protein and CD147 for preventing or treating meningoccal bacteraemia and/or infection. The present invention also relates to the combined use of such inhibitor and of an anti-bacterial compound, such as one used to prevent or treat a meningococcal infection. The invention also relates to a method for the prevention and/or treatment of meningococcal bacteraemia and/or infection, and to a method for screening inhibitors of the interaction between type IV pilus-associated protein and CD147.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. Ser. No.15/990,170, filed May 25, 2018, which was a divisional application ofU.S. Ser. No. 14/405,444, having a filing date of Dec. 4, 2014, whichwas a 371 application of International application PCT/EP2013/064900filed on Jul. 15, 2013, which claimed the benefit of European patentapplication 12305920.6, filed Jul. 27, 2012, all of said applicationsincorporated herein by reference.

FIELD OF THE INVENTION

The present invention concerns the use of an inhibitor of an interactionbetween type IV pilus-associated protein and CD147 for preventing ortreating meningococcal bacteraemia and/or infection. The presentinvention also relates to the combined use of such inhibitor and of ananti-bacterial compound, such as one used to prevent or treat ameningococcal infection. The invention also relates to a method for theprevention and/or treatment of meningococcal bacteraemia and/orinfection, and to a method for screening inhibitors of the interactionbetween type IV pilus-associated protein and CD147.

BACKGROUND TO THE INVENTION

Meningococcus (N. meningitidis, Nm), a cause of epidemic meningitis andsepsis, is a commensal Gram-negative bacterium of the human nasopharynx.After bloodstream invasion, virulent encapsulated bacteria adhere tobrain endothelial cells and proliferate onto the apical surface of hostcells to form microcolonies at the site of initial bacterial attachment,then cross the Blood Brain Barrier (BBB) to colonize meninges (Nassif etal., 2002, Trends Microbiol, 10:227-232).

The main clinical feature of meningococcal infection is meningitis.Paradoxically, Neisseria meningitidis is a frequent asymptomaticcolonizer of the human nasopharynx, and only a very small proportion ofinfections proceed to a sustained bacteraemia. In the majority ofbacteremic patients the presence of N. meningitidis in the bloodstreamwill remain asymptomatic. However during the blood phase, the bacteriawill interact with the brain endothelial cells and invade the meningesto be responsible for meningitis, which is the first clinicalcomplication of meningococcal dissemination in 70% of patients. In 30%of bacteremic patients the presence of meningococci in the bloodstreamwill lead to clinical symptoms of septicemia with in some cases apurpura fulminans. In the latter case the bacteria cross the blood brainbarrier; however, the meningitis symptoms are not at the forefront ofthe clinical presentation.

The emergence and epidemic potential of Nm are related to the expressionof outer membrane components (capsular polysaccharide andlipooligosaccharide [endotoxin]), while the propensity of Nm to interacttightly with peripheral and brain vascular cells relies on theexpression of type IV pili. These long filamentous structures, whichextend from the bacterial cell surface, exhibit astonishingmultifunctionality, including the attachment of virulent capsulatedmeningococci to cells and the triggering of host signalling events thatcontribute to the stabilization of bacterial colonies at the endothelialcell surface and promote subsequent translocation through endothelialbarriers. Type IV pili are composed of hetero-multimeric pilin subunits,which are assembled into helical fibers by a complex machinery. They arecomprised of the major pilin PilE required for piliation, and otherlow-abundance pilins (such as PilV, PilX or Comp) that structurallyresemble PilE and modulate the pilus adhesive and signalling functions.The adhesive properties of type IV pili are also modulated by componentsof the pilus machinery such as the PilC1 allele, making it difficult toidentify the molecular determinants involved in direct interaction withhost cell receptors using a genetic approach. As a consequence, theprecise adhesive components of the complex pilus structure remainundefined. However, it has been recently identified that the hostG-protein-coupled β2-adrenergic receptor is an essential endothelialreceptor for meningococcal type IV pili to trigger signalling events(Coureuil et al., 2010, Cell, 143:1149-1160; Lecuyer et al., 2011,Infect Immun, 80:175-186). Moreover, it has been identified that Nmcolonies at the cell surface of a human brain endothelial cell linepromote the translocation of βarrs to the inner surface of plasmamembrane, facing bacteria. βarrs translocated under the colonies serveas a scaffolding platform for signaling events elicited by Nm. Among theGPCRs expressed in the cell line, only the β2-adrenoceptor (β2AR) playsa permissive role in the formation of cortical plaques under coloniesand in bacterial crossing of cell monolayers. These observations revealthe requisition of a β2-adrenergic/β3-arrestin signaling pathway by Nmto promote stable adhesion onto human brain endothelial cells andsubsequent crossing of the BBB. More specifically, it has been shownthat interaction of β2AR, in particular of the N-terminus of β2AR, withtype IV pilus-associated proteins such as PilE or PilV, initiates theprocess allowing N. meningitidis to open and to traverse the BBB.

However, endothelial cells depleted of β2-adrenergic receptors stillsupport bacterial adhesion, indicating that type IV pili promote theprimary attachment of meningococci by interaction with another as yetunknown receptor.

Thus, preventing the primary attachment of meningococci to endothelialcells will prevent meningeal dissemination in the bloodstream, i.e.meningococcal bacteraemia, and/or meningococcal infection and, therebywill prevent bacterial meningitis and/or purpura fulminans.

SUMMARY OF THE INVENTION

The inventors have found that primary meningococcal adhesion toendothelial cells is mediated by the interaction between type IVpilus-associated proteins present at the surface of the meningococci andCD147 present at the surface of the host endothelial cells, and thatsuch interaction can be blocked, thereby inhibiting meningococcalbacteraemia, and consequently meningococcal infection.

Thus, the invention relates to an inhibitor of an interaction betweentype IV pilus-associated proteins and CD147 for use for preventing ortreating meningococcal bacteraemia and/or infection. Said inhibitor ispreferably (i) a CD147 inhibitor and/or (ii) a type IV pilus-associatedprotein inhibitor. Preferably, said interaction is a direct interaction.

In an embodiment, said inhibitor comprises a CD147 inhibitor which is apolypeptide able to interact with the CD147 receptor and therebyinhibits interaction between type IV pilus-associated protein and CD147receptor.

In an embodiment, this polypeptide is an anti-CD147 antibody or antibodyfragment, preferably directed against an extracellular portion of CD147,and by this interaction, in particular binding, said antibody inhibitsinteraction between type IV pilus-associated protein and CD147 receptoror prevent or abolish attachment of the type IV pilus-associated proteinto the CD147. In an embodiment, said anti-CD147 antibody is the MEM-M6/6antibody.

In another embodiment, the polypeptide may be a type IV pilus-associatedprotein fragment which is able to bind to the CD147 receptor and competewith the bacteria, thereby inhibiting interaction between type IVpilus-associated protein and CD147 receptor or prevent or abolishattachment of the type IV pilus-associated protein to the CD147.

In another embodiment, said inhibitor comprises a CD147 inhibitor whichis a nucleic acid, preferably a dsRNA which silences CD147 expression.Said CD147 inhibitor may comprise a siRNA or several siRNA silencingCD47 expression, preferably at least one siRNA of sequence SEQ ID NO: 1,2, 3, 4 or 5.

In another embodiment, said inhibitor comprises a type IVpilus-associated protein inhibitor which is a polypeptide able tointeract with the type IV pilus-associated protein and thereby inhibitsinteraction between type IV pilus-associated protein and CD147 receptor.

In an embodiment, this polypeptide is an anti-type IV pilus-associatedprotein antibody or antibody fragment, preferably directed against thebinding site to the CD147 receptor, and by this interaction, inparticular binding, said antibody inhibits interaction between type IVpilus-associated protein and CD147 receptor or prevents or abolishesattachment of the type IV pilus-associated protein to the CD147.

In another embodiment, this polypeptide is a CD147 polypeptide,preferably a polypeptide comprising CD147 amino acid sequence or afragment thereof able to bind to a type IV pilus-associated protein,wherein said antibody or polypeptide inhibits interaction between typeIV pilus-associated protein and CD147 receptor.

In an embodiment, said CD147 polypeptide comprises or consists of thesoluble form of the extracellular domain of CD147, or a fragment thereofwherein said soluble form or fragment inhibits interaction between typeIV pilus-associated protein and CD147 receptor. Said soluble form of theextracellular domain of CD147 may comprise or consist of the sequenceSEQ ID NO: 6, or a fragment thereof.

Also provided is a pharmaceutical composition comprising an inhibitor ofan interaction between type IV pilus-associated proteins and CD147 and apharmaceutically acceptable vehicle, diluent or carrier.

Also provided is a pharmaceutical composition comprising an inhibitor ofan interaction between type IV pilus-associated proteins and CD147 andadditionally at least one anti-bacterial compound. Said compositioncomprises a pharmaceutically acceptable vehicle, diluent or carrier.According to a feature, the composition is for a simultaneous, separateor sequential administration to a mammal, including human. Preferably,said at least one anti-bacterial compound is (i) a meningococcal vaccineantigen, or (ii) a fusion protein comprising at least two meningococcalvaccine antigens. Said meningococcal vaccine antigen may comprise orconsist of a meningococcal PilE, PilV, PilX, PilC, ComP, fHbp, PorA,NHBA, NadA, MafA, NspA, HmbR, TbpB, AusP, or a fragment thereof.

Further provided is the use of an inhibitor of an interaction betweentype IV pilus-associated proteins and CD147 in a method of preventingthe adhesion of a meningococcus to endothelial cells. This method may beused in vivo or in vitro.

Also provided is a method for preventing or treating meningococcalbacteraemia and/or meningococcal infection comprising administering toan individual in need thereof (mammal, preferably human) atherapeutically effective amount of an inhibitor of an interactionbetween type IV pilus-associated proteins and CD147, or a composition asdisclosed and provided herein.

Also provided is the use of an inhibitor of an interaction between typeIV pilus-associated proteins and CD147 for the manufacture of amedicament for preventing or treating meningococcal bacteraemia and/ormeningococcal infection.

Also provided is a method for screening inhibitors of the interactionbetween a type IV associated-pilus protein and the CD147 receptor,wherein said method comprises the step consisting of:

-   -   a) having a medium containing a type IV associated-pilus        protein, or a fragment thereof, and CD147 receptor, or a        fragment thereof, wherein said type IV associated-pilus protein,        or a fragment thereof, and said CD147 receptor, or a fragment        thereof, are able to specifically interact together to form a        binding pair,    -   b) contacting said medium with a candidate compound,    -   c) measuring the inhibition of the interaction between said type        IV associated-pilus protein, or a fragment thereof, and said        CD147 receptor, or a fragment thereof.

DEFINITIONS

By “meningococcal bacteraemia” is meant the transient passage of aviable meningococcus in the blood of the host without any clinicalsymptoms.

By “meningococcal infection” it is meant the sustained presence ofviable meningococcus in a host with clinical symptoms.

By “a meningococcus” is meant the Neisseria meningitidis (Nm) bacteria,preferably a Nm of serogroup A, B, C, Y or W135.

By “type IV pilus-associated protein”, is meant a protein that ispresent at the surface of the pilus of a bacterium. For example, saidtype IV pilus-associated protein may be the PilE, the PilV, the PilX,the PilC, or the ComP protein. Preferably, said type IV pilus-associatedprotein is a PilE or PilV protein, and more preferably a PilE or PilVprotein derived from any meningococcal serogroup, e.g. from ameningococcus of serogroup A, B, C, Y or W135.

By “CD147”, also called Basigin (BSG) or extracellular matrixmetalloproteinase inducer (EMMPRIN), is a member of the immunoglobulinsuperfamily that contains two heavily glycosylated C2 typeimmunoglobulin-like domains at the N-terminal extracellular portion. Insome embodiments, the CD147 comprises or consists of:

-   -   a) the sequence SEQ ID NO: 7 (NCBI Reference Sequence        NP_001719.2, update Feb. 26, 2012),    -   b) the sequence SEQ ID NO: 8 (NCBI Reference Sequence        NP_940991.1, update Feb. 26, 2012),    -   c) the sequence encoded by the nucleic acid SEQ ID NO: 9 (NCBI        Reference Sequence NM_001728.2, update Feb. 26, 2012),    -   d) the sequence encoded by the nucleic acid SEQ ID NO: 10 (NCBI        Reference Sequence NM_198589.1, update Feb. 26, 2012),    -   e) a sequence at least 80, 85, 90, 95, 96, 97, 98, 99% identical        to the sequence of a) to d).

By “interaction between type IV pilus-associated protein and CD147” ismeant the direct interaction between type IV pilus-associated proteinpresent at the surface of the meningococcus and CD147 present at thesurface of the endothelial cells, such as peripheral or brainendothelial cells, of the host. In fact, the inventors have found thatthe direct interaction between type IV pilus-associated protein andCD147 allows the adhesion of meningococcus to the endothelial cells,thereby allowing the meningococcus to infect blood vessels, such asbrain blood vessels and peripheral vessels.

By “inhibitor” is meant an agent that is able to reduce or to abolishthe interaction between type IV pilus-associated protein and CD147. Saidinhibitor may also be able to reduce or abolish the expression of CD147.According to the invention, said inhibitor is (i) a CD147 inhibitorand/or (ii) a type IV pilus-associated protein inhibitor.

Preferably, said inhibitor is able to reduce or to abolish theinteraction between type IV pilus-associated protein and CD147, by atleast 10, 20, 30, 40%, more preferably by at least 50, 60, 70%, and mostpreferably by at least 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100%.

Reference herein to polypeptides and nucleic acid includes both theamino acid sequences and nucleic acid sequences disclosed herein andvariants of said sequences.

Variant proteins may be naturally occurring variants, such as splicevariants, alleles and isoforms, or they may be produced by recombinantmeans. Variations in amino acid sequence may be introduced bysubstitution, deletion or insertion of one or more codons into thenucleic acid sequence encoding the protein that results in a change inthe amino acid sequence of the protein. Optionally the variation is bysubstitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more amino acids with any other amino acid in theprotein. Additionally or alternatively, the variation may be by additionor deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20 or more amino acids within the protein.

Variant nucleic acid sequences include sequences capable of specificallyhybridizing to the sequence of SEQ ID Nos: 1-5, 9, 10 under moderate orhigh stringency conditions. Stringent conditions or high stringencyconditions may be identified by those that: (1) employ low ionicstrength and high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5×SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mMsodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5×Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2×SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1×SSC containing EDTA at 55° C.Moderately stringent conditions may be identified as described bySambrook et al., Molecular Cloning: A Laboratory Manual, New York: ColdSpring Harbor Press, 1989, and include the use of washing solution andhybridization conditions (e.g., temperature, ionic strength and % SDS)less stringent that those described above. An example of moderatelystringent conditions is overnight incubation at 37° C. in a solutioncomprising: 20% formamide, 5×SSC (150 mM NaCl, 15 mM trisodium citrate),50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextransulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed bywashing the filters in 1×SSC at about 37-50° C.

Fragments of the proteins and variant proteins disclosed herein are alsoencompassed by the invention. Such fragments may be truncated at theN-terminus or C-terminus, or may lack internal residues, for example,when compared with a full length protein. Preferably, said fragments areat least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150,250, 300, 350, 400, 450, 500 or more amino acids in length.

Fragments of the nucleic acid sequences and variants disclosed hereinare also encompassed by the invention. Such fragments may be truncatedat 3′ or 5′ end, or may lack internal bases, for example, when comparedwith a full length nucleic acid sequence. Preferably, said fragments areat least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150,250, 300, 350, 400, 450, 500 or more bases in length.

Variant proteins may include proteins encoded by the variant nucleicacid sequences as described hereabove. Variant proteins may also includeproteins that have at least about 80% amino acid sequence identity witha polypeptide sequence disclosed herein. Preferably, a variant proteinwill have at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% amino acidsequence identity to a full-length polypeptide sequence or a fragment ofa polypeptide sequence as disclosed herein. Amino acid sequence identityis defined as the percentage of amino acid residues in the variantsequence that are identical with the amino acid residues in thereference sequence, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Sequence identity may be determined over the full length ofthe variant sequence, the full length of the reference sequence, orboth.

Variant nucleic acid sequences may include nucleic acid sequences thathave at least about 80% nucleotidic sequence identity with a nucleicacid sequence disclosed herein. Preferably, a variant nucleic acidsequences will have at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% aminoacid sequence identity to a full-length nucleic acid sequence or afragment of a nucleic acid sequence as disclosed herein. Nucleic acidsequence identity is defined as the percentage of nucleic acids in thevariant sequence that are identical with the nucleic acids in thereference sequence, after aligning the sequences and introducing gaps,if necessary, to achieve the maximum percent sequence identity, and notconsidering any conservative substitutions as part of the sequenceidentity. Sequence identity may be determined over the full length ofthe variant sequence, the full length of the reference sequence, orboth.

A peptide “substantially homologous” to a reference peptide may derivefrom the reference sequence by one or more conservative substitutions.Preferably, these homologous peptides do not include two cysteineresidues, so that cyclization is prevented. Two amino acid sequences are“substantially homologous” or “substantially similar” when one or moreamino acid residue are replaced by a biologically similar residue orwhen greater than 80% of the amino acids are identical, or greater thanabout 90%, preferably greater than about 95%, are similar (functionallyidentical).

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% (5 of 100) of the amino acidresidues in the subject sequence may be inserted, deleted, orsubstituted with another amino acid.

In the context of the present application, the percentage of identity iscalculated using a global alignment (i.e. the two sequences are comparedover their entire length). Methods for comparing the identity of two ormore sequences are well known in the art. The «needle » program, whichuses the Needleman-Wunsch global alignment algorithm (Needleman andWunsch, 1970 J. Mol. Biol. 48:443-453) to find the optimum alignment(including gaps) of two sequences when considering their entire length,may for example be used. The needle program is for example available onthe ebi.ac.uk world wide web site. The percentage of identity inaccordance with the invention is preferably calculated using theEMBOSS::needle (global) program with a “Gap Open” parameter equal to10.0, a “Gap Extend” parameter equal to 0.5, and a Blosum62 matrix.

Proteins consisting of an amino acid sequence “at least 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% identical” to a reference sequence maycomprise mutations such as deletions, insertions and/or substitutionscompared to the reference sequence. In case of substitutions, theprotein consisting of an amino acid sequence at least 80%, 85%, 90%,95%, 96%, 97%, 98% or 99% identical to a reference sequence maycorrespond to a homologous sequence derived from another species thanthe reference sequence.

Amino acid substitutions may be conservative or non-conservative.Preferably, substitutions are conservative substitutions, in which oneamino acid is substituted for another amino acid with similar structuraland/or chemical properties. The substitution preferably corresponds to aconservative substitution as indicated in the table below.

Conservative substitutions Type of Amino Acid Ala, Val, Leu, Ile, Met,Amino acids with aliphatic hydrophobic Pro, Phe, Trp side chains Ser,Tyr, Asn, Gln, Cys Amino acids with uncharged but polar side chains Asp,Glu Amino acids with acidic side chains Lys, Arg, His Amino acids withbasic side chains Gly Neutral side chain

The term “antibody” refers to immunoglobulin molecules andimmunologically active portions of immunoglobulin molecules, i.e.,molecules that contain an antigen binding site which immunospecificallybinds an antigen. As such, the term antibody encompasses intactmonoclonal antibodies, polyclonal antibodies, chimeric, humanized orhuman antibodies, antibodies, diabodies, multispecific antibodies (e.g.bispecific antibodies) formed from at least two intact antibodies,nanobodies, camelids antibodies, and also antibody fragments. In naturalantibodies, two heavy chains are linked to each other by disulfide bondsand each heavy chain is linked to a light chain by a disulfide bond.There are two types of light chain, lambda (λ) and kappa (κ). There arefive main heavy chain classes (or isotypes) which determine thefunctional activity of an antibody molecule: IgM, IgD, IgG, IgA and IgE.Each chain contains distinct sequence domains. The light chain includestwo domains, a variable domain (VL) and a constant domain (CL). Theheavy chain includes four domains, a variable domain (VH) and threeconstant domains (CH1, CH2 and CH3, collectively referred to as CH). Thevariable regions of both light (VL) and heavy (VH) chains determinebinding recognition and specificity to the antigen. The constant regiondomains of the light (CL) and heavy (CH) chains confer importantbiological properties such as antibody chain association, secretion,trans-placental mobility, complement binding, and binding to Fcreceptors (FcR). The Fv fragment is the N-terminal part of the Fabfragment of an immunoglobulin and consists of the variable portions ofone light chain and one heavy chain. The specificity of the antibodyresides in the structural complementarity between the antibody combiningsite and the antigenic determinant. Antibody combining sites are made upof residues that are primarily from the hypervariable or complementaritydetermining regions (CDRs). Occasionally, residues from nonhypervariable or framework regions (FR) influence the overall domainstructure and hence the combining site. Complementarity determiningregions (CDRs) refer to amino acid sequences which, together, define thebinding affinity and specificity of the natural Fv region of a nativeimmunoglobulin binding-site. The light and heavy chains of animmunoglobulin each have three CDRs, designated L-CDR1, L-CDR2, L-CDR3and H-CDR1, H-CDR2, H-CDR3, respectively. Therefore, an antigen-bindingsite includes six CDRs, comprising the CDR set from each of a heavy anda light chain V region.

Framework Regions (FRs) refer to amino acid sequences interposed betweenCDRs, i.e. to those portions of immunoglobulin light and heavy chainvariable regions that are relatively conserved among differentimmunoglobulins in a single species, as defined by Kabat, et al(Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md., 1991). As used herein, a “human framework region”is a framework region that is substantially identical (about 85%, ormore, in particular 90%, 95%, or 100%) to the framework region of anaturally occurring human antibody.

The term “monoclonal antibody” or “mAb” as used herein refers to anantibody molecule of a single amino acid composition, that is directedagainst a specific antigen and which may be produced by a single cloneof B cells or hybridoma. Monoclonal antibodies may also be recombinant,i.e. produced by protein engineering.

The term “chimeric antibody” refers to an engineered antibody whichcomprises a VH domain and a VL domain of an antibody derived from anon-human animal, in association with a CH domain and a CL domain ofanother antibody, in particular a human antibody. As the non-humananimal, any animal such as mouse, rat, hamster, rabbit or the like canbe used. A chimeric antibody may also denote a multispecific antibodyhaving specificity for at least two different antigens.

The term “humanized antibody” refers to antibodies in which theframework or “complementarity determining regions” (CDR) have beenmodified to comprise the CDR from a donor immunoglobulin of differentspecificity as compared to that of the parent immunoglobulin. In apreferred embodiment, a mouse CDR is grafted into the framework regionof a human antibody to prepare the “humanized antibody”.

“Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fv, Fab, F(ab′)2, Fab′,dsFv, scFv, sc(Fv)2, diabodies and multispecific antibodies formed fromantibody fragments.

The term “Fab” denotes an antibody fragment having a molecular weight ofabout 50,000 and antigen binding activity, in which about a half of theN-terminal side of H chain and the entire L chain, among fragmentsobtained by treating IgG with a protease, papaine, are bound togetherthrough a disulfide bond.

The term “F(ab′)₂” refers to an antibody fragment having a molecularweight of about 100,000 and antigen binding activity, which is slightlylarger than the Fab bound via a disulfide bond of the hinge region,among fragments obtained by treating IgG with a protease, pepsin.

The term “Fab′” refers to an antibody fragment having a molecular weightof about 50,000 and antigen binding activity, which is obtained bycutting a disulfide bond of the hinge region of the F(ab′)₂.

A single chain Fv (“scFv”) polypeptide is a covalently linked VH::VLheterodimer which is usually expressed from a gene fusion including VHand VL encoding genes linked by a peptide-encoding linker. The humanscFv fragment of the invention includes CDRs that are held inappropriate conformation, preferably by using gene recombinationtechniques. “dsFv” is a VH::VL heterodimer stabilised by a disulphidebond. Divalent and multivalent antibody fragments can form eitherspontaneously by association of monovalent scFvs, or can be generated bycoupling monovalent scFvs by a peptide linker, such as divalent sc(Fv)₂.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites.

By “antisense nucleic acid”, it is meant a non-enzymatic nucleic acidmolecule that binds to target RNA by means of RNA-RNA or RNA-DNA orRNA-PNA (protein nucleic acid; Egholm et al., 1993, Nature 365, 566)interactions and alters the activity of the target RNA (for a review,see Stein and Cheng, 1993, Science 261, 1004, and Woolf et al., U.S.Pat. No. 5,849,902). Typically, antisense molecules are complementary toa target sequence along a single contiguous sequence of the antisensemolecule. However, in certain embodiments, an antisense molecule canbind to substrate such that the substrate molecule forms a loop orhairpin, and/or an antisense molecule can bind such that the antisensemolecule forms a loop or hairpin. Thus, the antisense molecule can becomplementary to 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-contiguoussubstrate sequences or 2, 3, 4, 5, 6, 7, 8, 9, 10 or more non-contiguoussequence portions of an antisense molecule can be complementary to atarget sequence or both (for example, see Crooke, 2000, MethodsEnzymol., 313, 3-45). In addition, antisense DNA can be used to targetRNA by means of DNA-RNA interactions, thereby activating RNAse H, whichdigests the target RNA in the duplex. The antisense oligonucleotides cancomprise one or more RNAse H activating region, which is capable ofactivating RNAse H cleavage of a target RNA.

Upon introduction, the antisense nucleic acid enters a cellular pathwaythat is commonly referred to as the RNA interference (RNAi) pathway. Theterm “RNA interference” or “RNAi” refers to selective intracellulardegradation of RNA also referred to as gene silencing. RNAi alsoincludes translational repression by small interfering RNAs (siRNAs).RNAi can be initiated by introduction of Long double-stranded RNA(dsRNAs) or siRNAs or production of siRNAs intracellularly, eg from aplasmid or transgene, to silence the expression of one or more targetgenes. Alternatively RNAi occurs in cells naturally to remove foreignRNAs, eg viral RNAs. Natural RNAi proceeds via dicer directedfragmentation of precursor dsRNA which direct the degradation mechanismto other cognate RNA sequences.

In some embodiments, the antisense nucleic acid may be Longdouble-stranded RNAs (dsRNAs), microRNA (miRNA) and/or small interferentRNA (siRNA).

As used herein “Long double-stranded RNA” or “dsRNA” refers to anoligoribonucleotide or polyribonucleotide, modified or unmodified, andfragments or portions thereof, of genomic or synthetic origin or derivedfrom the expression of a vector, which may be partly or fully doublestranded and which may be blunt ended or contain a 5′ and or 3′overhang, and also may be of a hairpin form comprising a singleoligoribonucleotide which folds back upon itself to give a doublestranded region. In some embodiments, the dsRNA has a size ranging from150 bp to 3000 bp, preferably ranging from 250 bp to 2000 bp, still morepreferably ranging from 300 bp to 1000 bp. In some embodiments, saiddsRNA has a size of at least 150, 160, 170, 180, 190, 200, 210, 220,230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1500 bp. Insome embodiments, said dsRNA has a size of at most 3000, 2500, 2000,1500, 1000, 950, 900, 850, 800, 750,700, 650, 600, 550, 500, 450, 400,350, 300 bp.

A “small interfering RNA” or “siRNA” is a RNA duplex of nucleotides thatis targeted to a gene interest. A RNA duplex refers to the structureformed by the complementary pairing between two regions of a RNAmolecule. siRNA is targeted to a gene in that the nucleotide sequence ofthe duplex portion of the siRNA is complementary to a nucleotidesequence of the targeted gene. In some embodiments, the length of theduplex of siRNAs is ranging from 15 nucleotides to 50 nucleotides,preferably ranging from 20 nucleotides to 35 nucleotides, still morepreferably ranging from 21 nucleotides to 29 nucleotides. In someembodiments, the duplex can be of at least 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50nucleotides in length. In some embodiments, the duplex can be of at most45, 40, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20,19, 18, 17, 16, 15 nucleotides in length. The RNA duplex portion of thesiRNA can be part of a hairpin structure. In addition to the duplexportion, the hairpin structure may contain a loop portion positionedbetween the two sequences that form the duplex. The loop can vary inlength. In some embodiments the loop is 5, 6, 7, 8, 9, 10, 11, 12, or 13nucleotides in length. The hairpin structure can also contain 3 or 5overhang portions. In some embodiments, the overhang is a 3′ or a 5′overhang 0, 1, 2, 3, 4, or 5 nucleotides in length.

Injection and transfection antisense nucleic acid into cells andorganisms has been the main method of delivery. However, expressionvectors may also be used to continually express antisense nucleic acidin transiently and stably transfected mammalian cells. (See for example,e.g., Brummelkamp et al., 2002, Science, 296:550-553; Paddison et al.,2002, Genes & Dev, 16:948-958).

Antisense nucleic acid may be synthesized chemically or expressed viathe use of a single stranded DNA expression vector or equivalent thereofusing protocols known in the art as described for example in Carutherset al., 1992, Methods in Enzymology, 211:3-19; International PCTPublication No. WO 99/54459; Brennan et al., 1998, Biotechnol Bioeng,61:33-45; and U.S. Pat. No. 6,001,311. In a non-limiting example, smallscale syntheses are conducted on a 394 Applied Biosystems, Inc.synthesizer. Alternatively, the antisense nucleic acid molecules of thepresent invention can be synthesized separately and joined togetherpost-synthetically, for example by ligation (International PCTpublication No. WO 93/23569; Belton et al., 1997, Bioconjugate Chem,8:204).

The antisense nucleic acid of the invention may be able of decreasingthe expression of CD147, by at least 10, 20, 30, 40%, more preferably byat least 50, 60, 70%, and most preferably by at least 75, 80, 85, 90,95, 96, 97, 98, 99, 100%.

By “meningococcal vaccine antigen” is meant a meningococcal antigen thatis capable of raising an immune response against N. meningitidis, andthat is suitable for use as a vaccine or as an immunogenic compound.

By “immunogenic compound” is meant a compound that provokes orimmunomodulates immunosuppress or immunostimulate) an immune responsewhen administered to an individual or which induces the production ofantibodies when administered to an individual.

By “vaccine” as used herein refers to a compound, such as theimmunogenic compound described herein which is administered toimmunomodulate an immune response, that will protect or treat anindividual from illness, in particular due to that agent. The vaccinemay be a therapeutic (treatment) vaccine, i.e. for administration to theindividual after development of the disease with the intention to reduceor arrest disease progression, and/or a preventive (prophylactic)vaccine, for administration to the individual prior to the developmentof the disease, with the intent to prevent initial (and/or recurrent)infection.

The terms “subject”, “individual” or “host” are used interchangeably andmay be, for example, a human or a non-human mammal. Preferably, thesubject is a man, a woman, or a child.

Inhibitor of Interaction Between Type IV Pilus-Associated Protein andCD147

The present invention relates to an inhibitor of an interaction betweentype IV pilus-associated protein and CD147 receptor for use forpreventing or treating a meningococcal bacteraemia and/or infection,wherein said inhibitor is:

-   -   (i) a CD147 inhibitor, and/or    -   (ii) a type IV pilus-associated protein inhibitor.

In some embodiments, the CD147 inhibitor is an anti-CD147 antibody or anantisense nucleic acid, wherein said antibody inhibits interactionbetween type IV pilus-associated protein and CD147 receptor, and saidantisense nucleic acid silences CD147 receptor expression.

In one embodiment, said antisense nucleic acid may comprise or consistof a sequence that is able to inhibit or reduce the expression of aCD147 of sequence SEQ ID NO: 7 or 8, or a CD147 of sequence encoded bythe nucleic acid SEQ ID NO: 9 or 10. Said antisense nucleic acid maycomprise or consist of a sequence complementary to a nucleic acidencoding a CD147, for example a nucleic acid of sequence SEQ NO: 9 or10. Preferably, said antisense nucleic acid comprises or consists of asiRNA, in particular at least one siRNA of sequence SEQ ID NO: 1, 2, 3,4 or 5. In one embodiment, said siRNA comprises or consists of at least2, 3, 4 or 5 siRNA selected from the group consisting of SEQ ID NOs:1-5. In one embodiment, said siRNA comprises or consists of at most 5,4, 3, or 2 siRNA selected from the group consisting of SEQ ID NOs: 1-5.In one embodiment, said siRNA comprises or consists of the four siRNA ofsequence SEQ ID NO: 2, 3, 4 and 5.

Preferably, said anti-CD147 receptor antibody is able to prevent orinhibit the interaction between type-IV associated protein and CD147receptor, thereby preventing or inhibiting the adhesion of meningococcito endothelial cells, and consequently preventing or inhibitingmeningococcal bacteraemia and/or infection. Preferably, said anti-CD147antibody is an antibody directed against the binding site of CD147 totype IV pilus-associated proteins. Preferably, said anti-CD147 antibodyis directed against C-terminal domain of CD147 or to a fragment thereof.Still more preferably, said anti-CD147 antibody is directed to theectodomain of CD147 or to a fragment thereof. Accordingly, saidanti-CD147 is directed to the amino acids 16 to 321 of sequence SEQ IDNO: 7 or to a fragment thereof, or to the amino acids 16 to 205 ofsequence SEQ ID NO: 8, or to a fragment thereof. Preferably, saidanti-CD147 antibody is directed to a fragment of at least 10, 20, 30,40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100contiguous amino acids of said ectodomain of CD147. Still preferably,said anti-CD147 is directed to a fragment consisting of the amino acid221 to 315 of sequence SEQ ID NO:7 or to the amino acids 105 to 199 ofsequence SEQ ID NO:8, i.e. to the membrane proximal Immunoglobulindomain of CD147 (Ig-like V type domain). Still preferably, saidanti-CD147 antibody is the anti-CD147 antibody MEM-M6/6 (Abcam®).

In still another embodiment, the type IV pilus-associated proteininhibitor is an anti-type IV pilus-associated protein antibody.Preferably, said anti-type IV pilus-associated protein antibody is anantibody directed against the binding site of type IV pilus-associatedprotein to CD147 receptor, or to a fragment thereof. Still preferably,said anti-type IV pilus-associated protein antibody is an antibodydirected to the binding site of PilE and/or PilV protein to CD147receptor, or to a fragment thereof.

In still another embodiment, the type IV pilus-associated proteininhibitor is:

-   -   an immunogenic fragment of a type IV pilus-associated protein,        or    -   a fusion protein comprising or consisting of at least 2, 3, 4,        5, 6, 7, 8, 9, 10 immunogenic fragments of a type IV        pilus-associated protein,        wherein said immunogenic fragment or said fusion protein is        capable of raising an immune response with antibodies that        prevent the interaction between said type IV pilus-associated        protein and the CD147 receptor. Preferably, said immunogenic        fragment is an immunogenic fragment of PilE or PilV.        Consequently, said fusion protein may, for example, comprise or        consist of (i) at least 2, 3, 4, 5, 6, 7, 8, 9, 10 immunogenic        fragments of PilE, (ii) at least 2, 3, 4, 5, 6, 7, 8, 9, 10        immunogenic fragments of PilV, (iii) one immunogenic fragment of        PilE and one immunogenic fragment of PilV, (iv) 2 immunogenic        fragments of PilE and 2 immunogenic fragments of PilV, (v) one        immunogenic fragment of PilE and 2 immunogenic fragments of        PilV, etc.

In still another embodiment, the type IV pilus-associated proteininhibitor is a CD147 polypeptide. Preferably, said CD147 polypeptide isable to prevent or inhibit the interaction between said type-IVassociated protein and said CD147 receptor, thereby preventing orinhibiting the adhesion of meningococci to endothelial cells, andconsequently preventing or inhibiting meningococcal bacteraemia and/orinfection. Accordingly, said CD147 polypeptide may be a CD147 amino acidsequence that (i) differs from a complete CD147 of sequence SEQ ID NO: 7or 8, or from a complete CD147 of sequence encoded by the nucleic acidSEQ ID NO: 9 or 10, by one or several amino acid(s), and (ii) is notable to bind the endothelial cell membrane, or a fragment thereof.

In some embodiment, said CD147 polypeptide may comprise or consist ofthe extracellular domain of CD147, or a fragment thereof.

Preferably, said CD147 polypeptide comprises or consist of:

-   -   a) the amino acid sequence SEQ ID NO: 6, or    -   b) an amino acid sequence substantially homologous to SEQ ID        NO:6, preferably at least 80, 85, 90, 95, 96, 97, 98, or 99%        identical to SEQ ID NO:6.

For example, said fragment may comprise or consist of 10 to 180contiguous amino acid of said CD147 polypeptide, preferably 50 to 150amino acids, still preferably 80 to 120 amino acids. For example, saidfragment may comprise or consist of at least 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120,125, 130, 135, 140, 145, 150, 155, 160 ,165, 170 ,175, 180 contiguousamino acids of said CD147 polypeptide and/or at most 180, 175, 170, 165,160, 155, 150, 145, 140, 135, 130, 125, 120, 115, 110, 105, 100, 95, 90,85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10contiguous amino acids of said CD147 polypeptide.

Anti-Bacterial Compounds

In some embodiments, the inhibitor according to the invention is foradministration in combination with at least one anti-bacterial compound,either sequentially or simultaneously.

In some embodiments, the inhibitor according to the invention is foradministration in combination with at least 2, 3, 4, 5, 6, 7, 8, 9, 10and/or at most 10, 9, 8, 7, 6, 5, 4, 3, 2 anti-bacterial compounds,either sequentially or simultaneously.

Sequential administration indicates that the components are administeredat different times or time points, which may nonetheless be overlapping.Simultaneous administration indicates that the components areadministered at the same time.

The anti-bacterial compound may be a biological compound and/or achemical compound that inhibits or prevents development and/ormultiplication of bacteria. Preferably, said anti-bacterial compound isan anti-bacterial compound effective against Neisseria meningitidis(Nm), preferably a Nm of serogroup A, B, C, Y or W135.

The anti-bacterial compound may include, but is not limited to,antibiotics, such as the beta-lactamines antibiotics. Thebeta-lactamines antibiotics may be for example, but without limitation,(a) penicillines, such as benzylpenicilline, phenoxyméthylpenicilline,méticilline, dicloxacilline, flucloxacilline, amoxicilline, ampicilline,co-amoxiclav, pipéracilline, ticarcilline, azlocilline, carbénicilline;(b) cephalosporin, such as cephalexin, cephalotin, cephazolin, cefaclor,cefuroxim, cefamandole, cefotetan, cefoxitin, ceftriaxone, cefixime,cefotaxime, ceftazidime, cefepime, cefpirome; (c) carbapenemes, such asimipeneme, meropeneme, ertapeneme, doripeneme, aztreonam, clanulanicacid, tazobactam, sulbactam.

Preferably, said at least one anti-bacterial compound is (i) ameningococcal vaccine antigen, or (ii) a fusion protein comprising atleast two meningococcal vaccine antigens.

The meningococcal vaccine antigens according to the invention arewell-known to the skilled in the art. Indeed, several vaccines againstmeningococci are commercialized and/or under development.

The meningococcal vaccine antigen may comprise or consist of at leastone of the polysaccharide or glycoconjugate antigens comprised withinthe MCV-4 (commercialized under the name of Menactra® or Menveo®),MPSV-4 (Menomune®), Mencevax® or NmVac4-A/C/Y/W-135 vaccines.

The meningococcal vaccine antigen may also comprise or consists of thecapsule of group B (MenB), A, C, Y, and/or W135 meningococci and/or tothe outer membrane vesicle (OMV).

In some embodiments, the meningococcal vaccine antigen comprises orconsists of a polypeptide or polypeptides. Several such polypeptides orpolypeptidic antigens are currently investigated for their vaccinalpotency. The meningococcal vaccine antigen may be any of the proteins ofthe external surface of the meningococci. Thus the meningococcal vaccineantigen may be for example, the PilE protein (e.g. as described inPCT/EP2011/069463), the PilV protein (e.g. as described inPCT/EP2011/069463), the PilX protein, the PilC protein, the ComPprotein, Meningococcal Factor H binding protein (fHbp, previouslyreferred to as GNA1870 or Lp2086), the major immunodominant surfaceporin (PorA), Neisserial Heparin Binding Antigen (NHBA, previouslyreferred to as GNA2132), Neisseria adhesin A (NadA), MafA, Neisserialsurface protein A (NspA), Hemoglobin Receptor (HmbR),Transferrin-binding Protein B (TbPB), Autotransporter serine Protease(AusP), or fragments thereof. Preferably, said fragments are immunogenicfragments.

The PilE protein may derive from any meningococcal serogroup, e.g. froma meningococcus of serogroup A, B, C, Y or W135. For example, said PilEprotein may comprise or consist of the sequence of SEQ ID NO: 11 or SEQID NO: 12 or a sequence that is at least 80, 85, 90, 95 or 99% identicalto the sequence SEQ ID NO: 11 or SEQ ID NO: 12, or an immunogenicfragment thereof.

The PilV protein may derive from any meningococcal serogroup, e.g. froma meningococcus of serogroup A, B, C, Y or W135. For example, said PilVprotein may comprise or consist of the sequence of SEQ ID NO: 13 or asequence that is at least 80, 85, 90, 95 or 99% identical to thesequence SEQ ID NO: 13, or an immunogenic fragment thereof.

The PilX protein may derive from any meningococcal serogroup, e.g. froma meningococcus of serogroup A, B, C, Y or W135. For example, said PilXprotein may comprise or consist of the sequence of SEQ ID NO: 14 or asequence that is at least 80, 85, 90, 95 or 99% identical to thesequence SEQ ID NO: 14, or an immunogenic fragment thereof.

The PilC protein may derive from any meningococcal serogroup, e.g. froma meningococcus of serogroup A, B, C, Y or W135. For example, said PilCprotein may comprise or consist of the sequence of SEQ ID NO: 15 or asequence that is at least 80, 85, 90, 95 or 99% identical to thesequence SEQ ID NO: 15, or an immunogenic fragment thereof.

The ComP protein may derive from any meningococcal serogroup, e.g. froma meningococcus of serogroup A, B, C, Y or W135. For example, said ComPprotein may comprise or consist of the sequence of SEQ ID NO: 16 or asequence that is at least 80, 85, 90, 95 or 99% identical to thesequence SEQ ID NO: 16, or an immunogenic fragment thereof.

The fHBP protein may be a fHBP polypeptide of subfamilyB/Variant1 or ofsubfamilyA/Variant 2. For example, said fHBP polypeptide may comprise orconsist of the sequence SEQ ID NO: 17 or SEQ ID NO: 18 or a sequence atleast 80, 85, 90, 95 or 99% identical to the sequence SEQ ID NO: 17 orSEQ ID NO: 18, or an immunogenic fragment thereof.

The PorA protein may derive from any meningococcal serogroup, e.g. froma meningococcus of serogroup A, B, C, Y or W135. For example, said PorAprotein may comprise or consist of the sequence SEQ ID NO: 19 or asequence at least 80, 85, 90, 95 or 99% identical to the sequence SEQ IDNO: 19, or an immunogenic fragment thereof.

The NHBA protein may derive from any meningococcal serogroup, e.g. froma meningococcus of serogroup A, B, C, Y or W135. For example, said NHBAprotein may comprise or consist of the sequence SEQ ID NO: 20 or asequence at least 80, 85, 90, 95 or 99% identical to the sequence SEQ IDNO: 20, or an immunogenic fragment thereof.

The NadA protein may derive from any meningococcal serogroup, e.g. froma meningococcus of serogroup A, B, C, Y or W135. For example, said NadAprotein may comprise or consist of the sequence SEQ ID NO: 21 or asequence at least 80, 85, 90, 95 or 99% identical to the sequence SEQ IDNO: 21, or an immunogenic fragment thereof.

The MafA protein may derive from any meningococcal serogroup, e.g. froma meningococcus of serogroup A, B, C, Y or W135. For example, said MafAprotein may comprise or consist of the sequence of SEQ ID NO: 22, SEQ IDNO: 23, or SEQ ID NO: 24 or a sequence that is at least 80, 85, 90, 95or 99% identical to the sequence SEQ ID NO: 22, SEQ ID NO: 23, or SEQ IDNO: 24, or an immunogenic fragment thereof.

The NspA protein may derive from any meningococcal serogroup, e.g. froma meningococcus of serogroup A, B, C, Y or W135. For example, said NspAprotein may comprise or consist of the sequence of SEQ ID NO: 25 or asequence that is at least 80, 85, 90, 95 or 99% identical to thesequence SEQ ID NO: 25, or an immunogenic fragment thereof.

The HmbR protein may derive from any meningococcal serogroup, e.g. froma meningococcus of serogroup A, B, C, Y or W135. For example, said HmbRprotein may comprise or consist of the sequence of SEQ ID NO: 26 or asequence that is at least 80, 85, 90, 95 or 99% identical to thesequence SEQ ID NO: 26, or an immunogenic fragment thereof.

The TbpB protein may derive from any meningococcal serogroup, e.g. froma meningococcus of serogroup A, B, C, Y or W135. For example, said TbpBprotein may comprise or consist of the sequence of SEQ ID NO: 27 or asequence that is at least 80, 85, 90, 95 or 99% identical to thesequence SEQ ID NO: 27, or an immunogenic fragment thereof.

The AusP protein may derive from any meningococcal serogroup, e.g. froma meningococcus of serogroup A, B, C, Y or W135. For example, said AusPprotein may comprise or consist of the sequence of SEQ ID NO: 28 or asequence that is at least 80, 85, 90, 95 or 99% identical to thesequence SEQ ID NO: 28, or an immunogenic fragment thereof.

The immunogenic fragments according to the invention may be a contiguousportion of a meningococcal vaccine antigen as described herein that hasthe same or substantially the same immunogenic activity as saidmeningococcal vaccine antigen. That is to say, the fragment is capableof raising an immune response with antibodies that recognizes PilE,PilV, PilX, PilC, ComP, fHBP, NadA, PorA, NHBA, MafA, NspA, HmbR, TbPB,or AusP. Preferably, the fragment is capable of raising an immuneresponse with antibodies that prevent meningococcal infection. Morepreferably, said fragment is an immunogenic fragment of PilE or PilVthat is capable of raising an immune response with antibodies thatprevent meningococci to cross of the BBB or to spread into the meningealspace. Still more preferably, said fragment is an immunogenic fragmentof PilE or PilV that is capable of raising an immune response withantibodies that inhibit the interaction between PilE and/or PilV andβ2AR.

In some embodiment, said immunogenic fragment of PilE or PilV does notcarry the hydrophobic domain of PilE or PilV, respectively. Moreparticularly, said immunogenic fragment of PilE does not carry thehydrophobic domain of sequenceFTLIELMIVIAIVGILAAVALPAYQDYTARAQVSEAILLAEGQKSAVTEYYL (SEQ ID NO: 29).More particularly, said immunogenic fragment of PilV does not carry thehydrophobic domain of sequenceFTLLELMIAVAILGILTLITYPSYKTYIRRVRLSEVRTTLLHNAQTMERYYRQ (SEQ ID NO: 30).

In some embodiments, said immunogenic fragment of PilE comprises orconsists of:

-   -   a) the sequence        SAVTEYYLNHGEWPGDNSSAGVATSADIKGKYVQSVTVANGVITAQMASSNVNNEIKSKKLS        LWAKRQNGSVKWFCGQPVTRTTATATDVAAANGKTDDKINTKHLPSTCRDDSSAS (SEQ ID        NO:31), or    -   b) a fragment of at least 10, 15, 20, 25, 30, 35, 40, 45, 50,        55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115        consecutives amino acids of the sequence SEQ ID NO:31, or    -   c) a sequence that is at least 80, 85, 90, 95 or 99% identical        to the sequence of (a) or (b).

In some embodiments, said immunogenic fragment of PilV comprises orconsists of:

-   -   a) the sequence        TMERYYRQKGTFKTYDKNKLKQNKYFNVTLSKVSPDHFTLQADPNPTTNDGETCVVTLNDG        GTIAASGTNQSCPGFD (SEQ ID NO:32), or    -   b) a fragment of at least 10, 15, 20, 25, 30, 35, 40, 45, 50,        55, 60, 65, 70, 75 consecutives amino acids of the sequence SEQ        ID NO:32, or    -   c) a sequence that is at least 80, 85, 90, 95 or 99% identical        to the sequence of (a) or (b).

In some embodiments, said immunogenic fragment of PilE or PilV carriesthe recognition domain to the β2AR responsible for meningococcalsignaling.

More particularly, said immunogenic fragment of PilE comprises orconsists of:

-   -   a) the sequence CGQPVTRTTATATDVAAANGKTDDKINTKHLPSTC (SEQ ID NO:        33), or    -   b) a fragment of at least 10, 15, 20, 25, 30 consecutives amino        acids of the sequence SEQ ID NO:33, or    -   c) a sequence that is at least 80, 85, 90, 95 or 99% identical        to the sequence of (a) or (b).

More particularly, said immunogenic fragment of PilV comprises orconsists of:

-   -   a) the sequence GETCVVTLNDGGTIAASGTNQSCPGFD (SEQ ID NO:34), or    -   b) a fragment of at least 10, 15, 20, 25 consecutives amino        acids of the sequence SEQ ID NO :34, or    -   c) a sequence that is at least 80, 85, 90, 95 or 99% identical        to the sequence of (a) or (b).

In some embodiments, said fusion protein comprises at least 2, 3, 4, 5,6, 7, 8, 9, 10 or at most 10, 9, 8, 7, 6, 5, 4, 3, 2 meningococcalvaccine antigens as described herein.

For example, said fusion protein comprises or consists of:

-   -   a meningococcal PilE protein or an immunogenic fragment thereof,        fused in frame with a meningococcal PilV protein or an        immunogenic fragment thereof; or    -   a meningococcal PilE protein or an immunogenic fragment thereof,        fused in frame with a PilX, PilC, ComP, fHBP, NadA, PorA, NHBA,        MafA, NspA, HmbR, TbpB, or AusP protein or an immunogenic        fragment thereof; or    -   a meningococcal PilV protein or an immunogenic fragment thereof,        fused in frame with a PilX, PilC, ComP, fHBP, NadA, PorA, NHBA,        MafA, NspA, HmbR, TbpB, or AusP protein or an immunogenic        fragment thereof; or    -   a meningococcal PilE protein or an immunogenic fragment thereof,        fused in frame with (i) a meningococcal PilV protein or an        immunogenic fragment thereof, and (ii) a PilX, PilC, ComP, fHBP,        NadA, PorA, NHBA, MafA, NspA, HmbR, TbpB, or AusP protein or an        immunogenic fragment thereof.

For example, said fusion protein comprises or consists of theimmunogenic fragment of PilE of sequence SEQ ID NO: 31 fused with thefHBP polypeptide of sequence SEQ ID NO: 18, or of the immunogenicfragment of PilE of sequence SEQ ID NO: 33 fused with the fHBPpolypeptide of sequence SEQ ID NO: 18. Consequently, said fusion proteincomprises or consists of the sequence SEQ ID NO: 35 or SEQ ID NO: 36.

For example, said fusion protein comprises or consists of theimmunogenic fragment of PilV of sequence SEQ ID NO: 32 fused with thefHBP polypeptide of sequence SEQ ID NO: 18, or of the immunogenicfragment of PilV of sequence SEQ ID NO: 34 fused with the fHBPpolypeptide of sequence SEQ ID NO: 18. Consequently said fusion proteincomprises or consists of the sequence SEQ ID NO: 37 or SEQ ID NO: 38.

The fusion protein may further comprise other moieties such as e.g.peptidic linkers allowing linking the different antigens, proteins orfragments comprised within the fusion protein, a tag useful forpurification, a leader sequence, a signal peptide, one or more peptidicfragments enhancing the immunogenicity, etc.

Pharmaceutical Compositions

The invention also relates to a pharmaceutical composition comprising aninhibitor according to the invention and a pharmaceutical acceptablevehicle, diluent, or carrier.

Preferably, said inhibitor comprises or consists of:

-   -   (i) at least 1, 2, 3, 4 or 5, or at most 5, 4, 3, 2, or 1 siRNA        selected from the group consisting of siRNA of sequence SEQ ID        NOs: 1, 2, 3, 4 and 5 as defined hereabove,    -   (ii) the 4 siRNA of sequence SEQ ID NOs: 2, 3, 4, and 5 as        defined hereabove,    -   (iii) the anti-CD147 antibody MEM-M6/6 as defined hereabove, or    -   (iv) the variant CD147 of sequence SEQ ID NO: 6 as defined        hereabove.

In some embodiments, said pharmaceutical composition further comprisesat least one anti-bacterial compound. The active ingredients may bemixed in the same composition comprising a pharmaceutical acceptablevehicle, diluent, or carrier. They may be conditioned separately, forsimultaneous, separate or sequential administration to a mammal,including human. Said pharmaceutical composition may also comprise atleast 2, 3, 4, 5, 6, 7, 8, 9, 10 and/or at most 10, 9, 8, 7, 6, 5, 4, 3,2 anti-bacterial compounds.

Said at least one anti-bacterial compound may be a compound as definedabove. Preferably, said at least one anti-bacterial compound comprisesor consists of:

-   -   (a) at least a PilE, PilV, PilX, PilC, ComP, fHBP, NadA, PorA,        NHBA, MafA, NspA, HmbR, TbpB, or AusP protein or an immunogenic        fragment thereof as defined hereabove, or    -   (b) a fusion protein as defined hereabove.

In some embodiments, said pharmaceutical composition comprises:

-   -   the inhibitor (i) and the anti-bacterial compound (a),    -   the inhibitor (ii) and the anti-bacterial compound (a),    -   the inhibitor (iii) and the anti-bacterial compound (a),    -   the inhibitor (iv) and the anti-bacterial compound (a),    -   the inhibitor (i) and the anti-bacterial compound (b),    -   the inhibitor (ii) and the anti-bacterial compound (b),    -   the inhibitor (iii) and the anti-bacterial compound (b),    -   the inhibitor (iv) and the anti-bacterial compound (b),    -   the inhibitors (i) and (iii), and the anti-bacterial compound        (a),    -   the inhibitors (i) and (iv) and the anti-bacterial compound (a),    -   the inhibitors (i) and (iii), and the anti-bacterial compound        (b),    -   the inhibitors (i) and (iv) and the anti-bacterial compound (b),    -   the inhibitors (ii) and (iii), and the anti-bacterial compound        (a),    -   the inhibitors (ii) and (iv) and the anti-bacterial compound        (a),    -   the inhibitors (ii) and (iii), and the anti-bacterial compound        (b),    -   the inhibitors (ii) and (iv) and the anti-bacterial compound        (b),    -   the inhibitors (i), (iii) and (iv), and the anti-bacterial        compound (a),    -   the inhibitors (ii), (iii) and (iv), and the anti-bacterial        compound (a),    -   the inhibitors (i), (iii) and (iv), and the anti-bacterial        compound (b), or    -   the inhibitors (ii), (iii) and (iv), and the anti-bacterial        compound (b).

The pharmaceutical compositions according to the invention may beadministered orally in the form of a suitable pharmaceutical unit dosageform. The pharmaceutical compositions of the invention may be preparedin many forms that include tablets, hard or soft capsules, especiallyhard or soft gelatin capsules, aqueous solutions, suspensions, andliposomes and other slow-release formulations, such as shaped polymericgels.

The mode of administration and dosage forms are closely related to theproperties of the therapeutic agents or compositions which are desirableand efficacious for the given treatment application. Suitable dosageforms include, but are not limited to, oral, intravenous, rectal,sublingual, mucosal, nasal, ophthalmic, subcutaneous, intramuscular,transdermal, spinal, intrathecal, intra-articular, intra-arterial,sub-arachnoid, bronchial, and lymphatic administration, and other dosageforms for systemic delivery of active ingredients.

Pharmaceutical compositions of the invention may be administered by anymethod known in the art, including, without limitation, transdermal(passive via patch, gel, cream, ointment or iontophoretic); intravenous(bolus, infusion); subcutaneous (infusion, depot); transmucosal (buccaland sublingual, e.g., orodispersible tablets, wafers, film, andeffervescent formulations; conjunctival (eye drops); rectal(suppository, enema)); or intradermal (bolus, infusion, depot).

The solid unit dosage forms can be of the conventional type. The solidform can be a capsule, such as an ordinary gelatin type containing theinhibitor of the present invention, and optionnally an anti-bacterialcompound of the present invention, and a carrier, for example,lubricants and inert fillers such as, lactose, sucrose, or cornstarch.In another embodiment, these compounds are tableted with conventionaltablet bases such as lactose, sucrose, or corn starch in combinationwith binders like acacia, corn starch, or gelatin, disintegrating agentssuch as, corn starch, potato starch, or alginic acid, and a lubricantlike stearic acid or magnesium stearate.

Oral liquid pharmaceutical compositions may be in the form of, forexample, aqueous or oily suspensions, solutions, emulsions, syrups orelixirs, or may be presented as a dry product for constitution withwater or other suitable vehicle before use. Such liquid pharmaceuticalcompositions may contain conventional additives such as suspendingagents, emulsifying agents, non-aqueous vehicles (which may includeedible oils), or preservatives.

Pharmaceutical compositions of the invention may also be formulated forparenteral administration (e.g., by injection, for example, bolusinjection or continuous infusion) and may be presented in unit dosageform in ampoules, pre-filled syringes, small volume infusion containersor multi-dose containers with an added preservative. The pharmaceuticalcompositions may take such forms as suspensions, solutions, or emulsionsin oily or aqueous vehicles, and may contain formulating agents such assuspending, stabilizing and/or dispersing agents. Alternatively, thepharmaceutical compositions of the invention may be in powder form,obtained by aseptic isolation of sterile solid or by lyophilization fromsolution, for constitution with a suitable vehicle, e.g. sterile,pyrogen-free water, before use.

Pharmaceutical compositions suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art, and the suppositories may beconveniently formed by admixture of the pharmaceutical composition withthe softened or melted carrier(s) followed by chilling and shaping inmolds.

For administration by inhalation, the pharmaceutical compositionsaccording to the invention are conveniently delivered from aninsufflator, nebulizer or a pressurized pack or other convenient meansof delivering an aerosol spray. Pressurized packs may comprise suitablepropellant such as dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Alternatively, foradministration by inhalation or insufflation, the pharmaceuticalcompositions of the invention may take the form of a dry powdercomposition, for example, a powder mix of the pharmaceutical compositionand a suitable powder base such as lactose or starch. The powdercomposition may be presented in unit dosage form in, for example,capsules or cartridges or, e.g., gelatin or blister packs from which thepowder may be administered with the aid of an inhalator or insufflator.

For intra-nasal administration, the pharmaceutical compositions of theinvention may be administered via a liquid spray, such as via a plasticbottle atomizer. Typical of these are the Mistometerg (isoproterenolinhaler-Wintrop) and the Medihaler® (isoproterenol inhaler-Riker).

For antisense nucleic acid administration, the pharmaceuticalcompositions of the invention may be prepared in forms that includeencapsulation in liposomes, microparticles, microcapsules, lipid-basedcarrier systems. Non limiting examples of alternative lipid basedcarrier systems suitable for use in the present invention includepolycationic polymer nucleic acid complexes (see, e.g. US PatentPublication No 20050222064), cyclodextrin polymer nucleic acid complexes(see, e.g. US Patent Publication No 20040087024), biodegradable poly 3amino ester polymer nucleic acid complexes (see, e.g. US PatentPublication No 20040071654), pH sensitive liposomes (see, e.g. US PatentPublication No 20020192274), anionic liposomes (see, e.g. US PatentPublication No 20030026831), cationic liposomes (see, e.g. US PatentPublication No 20030229040), reversibly masked lipoplexes (see, e.g. USPatent Publication No 20030180950), cell type specific liposomes (see,e.g. US Patent Publication No 20030198664), microparticles containingpolymeric matrices (see, e.g. US Patent Publication No 20040142475), pHsensitive lipoplexes (see, e.g. US Patent Publication No 20020192275),liposomes containing lipids derivatized with releasable hydrophilicpolymers (see, e.g. US Patent Publication No 20030031704), lipid entrapped nucleic acid (see, e.g. PCT Patent Publication No WO 03/057190),lipid encapsulated nucleic acid (see, e.g. US Patent Publication No20030129221), polycationic sterol derivative nucleic acid complexes(see, e.g. US Pat No 6,756,054), other liposomal compositions (see, e.g.US Patent Publication No 20030035829), other microparticle compositions(see, e.g. US Patent Publication No 20030157030), poly-plexes (see, e.g.PCT Patent Publication No WO 03/066069), emulsion compositions (see,e.g. U.S. Pat. No. 6,747,014), condensed nucleic acid complexes (see,e.g. US Patent Publication No 20050123600), other polycationic nucleicacid complexes (see, e.g. US Patent Publication No 20030125281),polyvinylether nucleic acid complexes (see, e.g. US Patent PublicationNo 20040156909), polycyclic amidinium nucleic acid complexes (see, e.g.US Patent Publication No 20030220289), nanocapsule and microcapsulecompositions (see, e.g. PCT Patent Publication No WO 02/096551),stabilized mixtures of liposomes and emulsions (see, e.g. EP1304160),porphyrin nucleic acid complexes (see, e.g. U.S. Pat. No. 6,620,805),lipid nucleic acid complexes (see, e.g. US Patent Publication No20030203865), nucleic acid micro emulsions (see, e.g. US PatentPublication No 20050037086), and cationic lipid based compositions (see,e.g. US Patent Publication No 20050234232). One skilled in the art willappreciate that modified siRNA of the present invention can also bedelivered as a naked siRNA molecule.

Pharmaceutical compositions of the invention may also contain otheradjuvants such as flavorings, colorings, or preservatives.

It will be further appreciated that the amount of the pharmaceuticalcompositions required for use in treatment will vary not only with thetherapeutic agent selected but also with the route of administration,the nature of the condition being treated and the age and condition ofthe patient and will be ultimately at the discretion of the attendantphysician or clinician.

Method for Preventing Adhesion of Meningococci to an Endothelial Cell

The inhibitor according to the invention may be used in a method ofpreventing or inhibiting adhesion of meningococci to an endothelialcell.

Said method may be an in vitro or ex vivo method.

The invention thus provides the use of an inhibitor as defined herein inan in vitro or in vivo method for preventing or inhibiting adhesion ofmeningococci to an endothelial cell.

In some embodiments, said inhibitor is used in combination with at leastone anti-bacterial compound as defined hereabove. In some embodiments,said inhibitor is used in combination with at least 2, 3, 4, 5, 6, 7, 8,9, 10 and/or at most 10, 9, 8, 7, 6, 5, 4, 3, 2 anti-bacterial compoundsas defined hereabove.

Said method may comprise, for example, exposing said cell and/or saidmeningococcus to said inhibitor. Where the method is an in vivo method,the method may comprise administering said inhibitor to a subject,preferably a patient in need thereof.

In some embodiments, said endothelial cells may be brain endothelialcells, capillaries endothelial cells, human dermal endothelial cellsand/or umbilical vein endothelial cells.

Administration and Methods of Treatment

The invention also relates to a method for preventing or treatingmeningococcal bacteraemia or infection in an individual in need thereofcomprising administering a therapeutically effective amount of aninhibitor according to the invention or a pharmaceutical composition ofthe invention.

By “treatment” is meant a therapeutic use (i.e. on a patient having agiven disease) and by “preventing” is meant a prophylactic use (i.e. onan individual susceptible of developing a given disease). The term“treatment” not only includes treatment leading to complete cure of thedisease, but also treatments slowing down the progression of the diseaseand/or prolonging the survival of the patient.

An “effective amount” refers to an amount effective, at dosages and forperiods of time necessary, to achieve the desired therapeutic orprophylactic result.

A therapeutically effective amount of an inhibitor of the invention mayvary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the protein, to elicit adesired therapeutic result. A therapeutically effective amountencompasses an amount in which any toxic or detrimental effects of theinhibitor are outweighed by the therapeutically beneficial effects. Atherapeutically effective amount also encompasses an amount sufficientto confer benefit, e.g., clinical benefit.

In the context of the present invention, “preventing a meningococcalbacteraemia or infection” may mean prevention of meningococcus adhesionto the endothelial cells of the host.

In the context of the present invention, “treating a meningococcalbacteraemia or infection”, may mean reversing, alleviating, orinhibiting meningococcus adhesion to the endothelial cells of the host.

In the context of the invention, meningococcal infection may be reducedby at least 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 85, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100%.

In some embodiments, the methods of the invention comprise theadministration of an inhibitor as defined above, in combination with atleast one anti-bacterial compound as defined above, either sequentiallyor simultaneously. For example, said at least other anti-bacterialcompound is (i) a meningococcal vaccine antigen, or (ii) a fusionprotein comprising at least two meningococcal vaccine antigens, asdescribed hereabove.

In another embodiment, said method comprises the administration of apharmaceutical composition according to the invention.

The administration regimen may be a systemic regimen. The mode ofadministration and dosage forms are closely related to the properties ofthe therapeutic agents or compositions which are desirable andefficacious for the given treatment application. Suitable dosage formsand routes of administration include, but are not limited to, oral,intravenous, rectal, sublingual, mucosal, nasal, ophthalmic,subcutaneous, intramuscular, transdermal, spinal, intrathecal,intra-articular, intra-arterial, sub-arachnoid, bronchial, and lymphaticadministration, and/or other dosage forms and routes of administrationfor systemic delivery of active ingredients. In a preferred embodiment,the dosage forms are for parenteral administration.

The administration regimen may be for instance for a period of at least5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100 days.

The dose range may be between 0.1 mg/kg/day and 100 mg/kg/day. Morepreferably, the dose range is between 0.5 mg/kg/day and 100 mg/kg/day.Most preferably, the dose range is between 1 mg/kg/day and 80 mg/kg/day.Most preferably, the dose range is between 5 mg/kg/day and 50 mg/kg/day,or between 10 mg/kg/day and 40 mg/kg/day.

In some embodiments, the dose may be of at least 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6, 7, 8, 9,10, 15, 20, 25, 30, 35, 40, 45, 50 mg/kg/day. In some embodiments, thedose may be of at most 50, 45, 40, 35, 30, 25, 20, 25, 15, 10, 5, 1,0.5, 0.1 mg/kg/day.

The dose range may also be between 10 to 10000 UI/kg/day. Morepreferably, the dose range is between 50 to 5000 UI/kg/day, or between100 to 1000 UI/kg/day.

In some embodiments, the dose may be of at least 10, 25, 50, 75, 100,150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 1500,2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500,8000, 8500, 9000, 9500, 10000 UI/kg/day. In some embodiments, the dosemay be of at most 10000, 9500, 9000, 8500, 8000, 7500, 7000, 6500, 6000,5500, 5000, 4500, 4000, 3500, 3000, 2500, 2000, 1500, 1000, 900, 800,600, 500, 450, 400, 350, 300, 250, 200, 150, 100 UI/kg/day.

Screening Method

The invention also relates to a method for screening inhibitors of theinteraction between a type IV associated-pilus protein and the CD147receptor, wherein said method comprises the step consisting of:

-   -   a) having a medium containing a type IV associated-pilus        protein, or a fragment thereof, and CD147 receptor, or a        fragment thereof, wherein said type IV associated-pilus protein,        or a fragment thereof, and said CD147 receptor, or a fragment        thereof, are able to specifically interact together to form a        binding pair,    -   b) contacting said medium with a candidate compound,    -   c) measuring the inhibition of the interaction between said type        IV associated-pilus protein, or a fragment thereof, and said        CD147 receptor, or a fragment thereof.

Preferably, step b) is performed by adding the candidate compound to themedium of step a).

Compounds that inhibits the interaction are selected. This selection maybe performed if the measuring in step c) demonstrates a significantinhibition of said interaction.

In some embodiments, said type IV associated-pilus protein comprises orconsists of a PilE or PilV protein or to a fragment thereof that is ableto interact with CD147 receptor.

In some embodiments, said fragment of CD147 receptor comprises orconsists of the ectodomain of CD147 or to the membrane proximalImmunoglobulin domain of CD147, or to the extracellular domain of CD147of sequence SEQ ID NO: 6.

In some embodiments, the method according to the present invention ischaracterized in that at step c) the measure of the inhibition of theinteraction is carried out by immunoassay (particularly by ELISA or byImmunoradiometric Assay (IRMA)), by Scintillation Proximity Assay (SPA)or by Fluorescence Resonance Energy Transfer (FRET).

Preferably, the method according to the present invention method isbased on the Alphascreen® technology. Alphascreen® is based on Donor (D,photosensitizer) and Acceptor (A, chemiluminescer) microbeads, coatedwith two molecules of interest, susceptible of binding to each other.Laser excitation of the D beads causes ambient oxygen to be converted tothe singlet state by the photosensitizer. Singlet oxygen species in turnactivate chemiluminescent agents on the A beads. Upon activation, thechemiluminescent agent emits light which is detected by thephoto-detector in a microplate reader. A signal is produced when the Aand D beads are brought into proximity (<200 nm) thus reporting for theinteraction between the partners captured on the two beads (Taouji etal., 2009, Curr Genomics, 10(2):93).

Accordingly, in an embodiment, at step a) said type IV associated-pilusprotein, or fragment thereof, is bound to an acceptor bead and saidCD147 receptor, or fragment thereof, is bound to a donor bead. Inanother embodiment, at step a) said type IV associated-pilus protein, orfragment thereof, is bound to a donor bead and said CD147 receptor, orfragment thereof, is bound to an acceptor bead. Said type IVassociated-pilus protein, or fragment thereof, and CD147, or fragmentthereof, may be directly or indirectly bound to said beads.

Step a) is performed in conditions that allow the interaction betweensaid type IV associated-pilus protein and said CD147 receptor.Accordingly, the binding of said type IV associated-pilus protein andsaid CD147 receptor will result in bringing the donor and acceptorparticle at a distance less than 200 nm from each other, therebyproviding an energy transfer between the donor and acceptor molecules.

In some embodiments, the donor bead contains phtalocyanine and theacceptor bead contains rubren. The donor bead is submitted to a laserexcitation at 680 nm, inducing the conversion of ambient oxygen to asinglet state oxygen. The singlet state oxygen reacts with thechemiluminescent agent, i.e. rubren, on the acceptor bead. Upon energytransfer, activated rubrene emits light at 520-620 which is detected bythe photodetector in a microplate reader. The luminescence can forexample be measured with a photodetector in a microwell plate reader,such as the EnVision® multilabel plate reader from Perkin-Elmer.

In some embodiments, the donor and acceptor beads are bound tofluorophores and the energy transfer amounts to fluorescence is emittedupon excitation by a first donor fluorophore, towards a second acceptorfluorophore. The acceptor fluorophore is, thus, activated and emits asecond fluorescence. The fluorescence of the second acceptor fluorophorecan be measured for example by a spectrofluorometer.

If energy transfer is detected at step a), it is considered that thereis an interaction between said type IV associated-pilus protein, orfragment thereof, and said CD147 receptor, or fragment thereof.

The candidate compounds include but are not limited to peptides, smallmolecules, antibodies, aptamers and nucleic acids such as antisensenucleic acids.

Measuring the inhibition of interaction between said type IVpilus-associated protein, or fragment thereof, and said CD147 receptor,or fragment thereof, at step c) may be performed by measuring theluminescence or the fluorescence. If the level of luminescence orfluorescence at step c) is lower than the level of luminescence orfluorescence at step a), it indicates that the candidate compound is aninhibitor of the interaction of said type IV associated-pilus protein,or fragment thereof, and said CD147 receptor, or fragment thereof.

The invention will now be described in more detail with reference to thefollowing figures and examples. All literature and patent documentscited herein are hereby incorporated by reference.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Identification of CD147 as N. meningitidis receptor candidate.TPA treatment induces Nm adhesion to BB19 cells: cells were untreated(white) or treated with TPA (10 ng/ml, black) in the absence (−) or inthe presence of Actinomycin D (1 μg/ml). Adhesion of wild typemeningococci (WT) was measured 48 h after treatment. Mean±s.e.m,n>_3;***P<0.0001; Two-way Anova.

FIG. 2. Identification of CD147 as N. meningitidis receptor candidate.TPA treatment induces Nm adhesion to BB19 cells: cells were untreated(white) or treated with TPA (10 ng/ml, black). Adhesion of wild typemeningococci (WT) or of the bacterial mutants dpilE, dpilC1 or dpilC2was measured 48 h after treatment. Mean±s.e.m, n>_3;***P<0.0001; Two-wayAnova.

FIG. 3. CD147 accumulates at sites of bacterial adhesion independentlyof the 02-adrenergic receptor-coupled signalling events.Isoproterenol-induced β2-adrenergic receptor endocytosis inhibited typeIV pilus-induced signaling events, but did not affect CD147 accumulationat sites of bacterial adhesion. Quantification of Ezrin, Actin, CD44 orCD147 recruitment at sites of Nm adhesion upon cell pretreatement with10 IaM isoproterenol. Mean±s.e.m, n=3;***P<0.001; **P<0.01; NSP>0.05;Two-way Anova.

FIG. 4. CD147 knockdown reduced Nm adhesion to hCMEC/D3s relative tocontrol siRNA (CTL). Mean±s.e.m, n=4;**P<0.05; Two-tailed t test.

FIG. 5. Expression of exogenous CD147 restored bacterial adhesion toCD147 knock-down hBMEC cells. Mean±s.e.m, n=3;**P=0.0025; One-way Anova.

FIG. 6. CD147 knockdown reduced the initial attachment of Nm to hBMECcells under shear stress. Mean±s.e.m, n=3;**P<0.01. One-way Anova.

FIG. 7. CD147-Fc but not ICAM-1-Fc (both 5 μg/ml) reduced Nm adhesion tohBMEC cells. Mean±s.e.m, n=3;**P<0.01; One-way Anova.

FIG. 8. AntiCD147 MEM-M6/6 [10 μg/ml] potently inhibited Nm adhesion tohBMEC cells, other antibodies [10 μg/ml] scarcely (MEM-M6/1) or did not(anti-ICAM-1, 11C81) affect adhesion. Mean±s.e.m, n=3;***P<0.001;**P<0.001; One-way Anova.

FIG. 9. Nm directly adhere on immobilised CD147-Fc, but not ICAM-1-Fc.Negative: no immobilised proteins. Bar: mean per field. Mean±s.e.m,n=3;***P<0.001; One-way Anova.

FIG. 10. Wild type (WT), dpilE, dpilC1 or dpilC2 meningococci adheringto immobilised CD147-Fc. Bar: mean per field. Mean±s.e.m,n=4;***P<0.001; One-way Anova.

FIG. 11. Purified MBP-PilE and MBP-PilV chimera interact directly withCD147-Fc, whereas MBP-PilX, MBP-ComP and MBP did not. Top: CD147-Fcco-precipitating with staphylococci carrying the MBP-Pilins indicated.Bottom: Quantification of bound CD147-Fc. Mean±s.e.m,n=4;***P<0.001;**P<0.01; One-way Anova.

FIG. 12. Soluble MBP-PilE and MBP-PilV potently inhibited bacterialadhesion to hCMEC/D3 cells, whereas MBP-PilX, MBP-ComP and MBP wereineffective. Mean±s.e.m, n=4;***P<0.001; **P<0.01; One-way Anova.

FIG. 13. Bacterial mutants ΔpilE andΔApilV failed to adhere to hCMEC/D3cells. Mean±s.e.m, n=4;***P<0.001; One-way Anova.

FIG. 14. Quantification of adherent fluorescent meningococci/100 μm2section, expressed as % of adherent WT. Mean±s.e.m n=3, brain sectionsfrom 3 different donors, ***P<0.001; One-way Anova.

FIG. 15. In Situ infection of fresh human brain sections by N.meningitidis required pilus interaction with CD147. Mean±s.e.m of totalfluorescence/100 μm2 relative to fluorescence in absence of antibody,n=4, brain sections from 3 different donors, *P<0.05; One-way Anova.

FIG. 16. The pilus of Neisseria meningitidis is required for targetinghuman skin xenografts if SCID mice (Intraperitoneal route, 106 CFUs, 3mice per group).

FIG. 17. Impact of human skin xenograft on bacteraemia in SCID mice(Intraveinous route, 10⁵ CFUs, 3 mice per group).

FIG. 18. Survival of Neisseria meningitidis infected SCID mice(Intraveinous route, 10⁵ CFUs, 3 mice per group).

BRIEF DESCRIPTION OF THE SEQUENCE LISTING

SEQ ID NOs: 1 to 5 show the sequences of the siRNA against CD147.

SEQ ID NO: 6 shows the sequence of the extracellular domain of CD147.

SEQ ID NO: 7 shows the amino acid sequence of CD147 referenced under theNCBI Reference Sequence Number NP_001719.2.

SEQ ID NO: 8 shows the amino acid sequence of CD147 referenced under theNCBI Reference Sequence Number NP_940991.1.

SEQ ID NO: 9 shows the nucleotidic sequence of CD147 referenced underthe NCBI Reference Sequence Number NM_001728.2.

SEQ ID NO: 10 shows the nucleotidic sequence of CD147 referenced underthe NCBI Reference Sequence Number NM_198589.1.

SEQ ID NOs: 11 and 12 show amino acid sequences of PilE.

SEQ ID NO: 13 shows the amino acid sequence of PilV.

SEQ ID NO: 14 shows the amino acid sequence of PilX.

SEQ ID NO: 15 shows the amino acid sequence of PilC.

SEQ ID NO: 16 shows the amino acid sequence of ComP.

SEQ ID NOs: 17 and 18 show amino acid sequences of fHBP.

SEQ ID NO: 19 shows the amino acid sequence of PorA.

SEQ ID NO: 20 shows the amino acid sequence of NHBA.

SEQ ID NO: 21 shows the amino acid sequence of NadA.

SEQ ID NOs: 22 to 24 show amino acid sequences of MafA.

SEQ ID NO: 25 shows the amino acid sequence of NspA.

SEQ ID NO: 26 shows the amino acid sequence of HmbR.

SEQ ID NO: 27 shows the amino acid sequence of TbpB.

SEQ ID NO: 28 shows the amino acid sequence of AusP.

SEQ ID NO: 29 shows the sequence of the hydrophobic domain of PilE.

SEQ ID NO: 30 shows the sequence of the hydrophobic domain of PilV.

SEQ ID NOs: 31 and 33 show the sequences of immunogenic fragments ofPilE.

SEQ ID NOs: 32 and 34 show the sequences of immunogenic fragments ofPilV.

SEQ ID NOs: 35 and 36 show the sequences of the fusion proteins betweenPilE and fHBP polypeptides.

SEQ ID NOs: 37 and 38 show the sequences of the fusion proteins betweenPilV and fHBP polypeptides.

SEQ ID NO: 39 shows the sequence of a control siRNA.

EXAMPLES Material and Methods Serial analysis of Gene Expression Adaptedfor Downsized Extracts (SADE)

200 μg of RNA extracted from 2×107 untreated or TPA treated BB19 cells16 hours after treatment with TPA, was used as a substrate for SADE. TheSADE screen was carried out as described in Virlon et al., 1999, ProcNatl Acad Sci USA, 96:15286-15291. Briefly, total RNA was extracted fromcells, poly-adenylated RNA was selected on oligo-dT columns and taggedcDNA was synthesized from the poly-A RNAs. Concatemers of DNA tags weresequenced and the number of sequenced tags differentially represented inthe two libraries was determined and analysed using Monte Carlostatistical analysis.

Antibodies and Reagents

Anti-hCD147 antibodies were purchased from AbD Serotec, anti-hCD44 mAbfrom Immunotech, antihlCAM-1 from R&D Systems, anti-GFAP andanti-vimentin from Sigma Aldrich. Polyclonal antiserum raised againstezrin was obtained from P. Mangeat (CNRS UMR 5539, Montpelier, France).mAb raised against PilE (20D9) and polyclonal antiserum raised againstmeningococcal 2C4.3 strain were described previously (Coureuil et al.,2010, Cell, 143:1149-1160). Secondary antibodies used forimmunofluorescence labeling, chromogenic immunohistochemistry andimmunoblotting were from Jackson ImmunoResearch Laboratories. Solublechimeric CD147-Fc and ICAM-1-Fc molecules were purchased from R&DSystems. DAPI, Rhoda mine-phalloidin, TPA, Actinomycin D, Isoproterenol,DAB Peroxidase Substrate and NBT/BCIP phosphatase alkaline substratewere purchased from Sigma Aldrich.

Cell Culture, Transfection and Infection

BB19, a human brain endothelial cell line transformed by papilloma viruswas kindly provided by Dr J A. Nelson (Oregon Health and ScienceUniversity, Beaverton, USA) (Prudhomme et al., 1996, Int J Parasitol,26:647-655). To induce bacterial adhesion, BB19 were treated with TPA(10 ng/ml, for 10′), washed and 48 h after treatment, meningococcaladhesion was measured. When indicated 2 h after TPA treatment thetranscriptional inhibitor Actinomycin D was added (1 μg/ml, for 4 h),cells were washed and adhesion was measured 42 h later. The hCMEC/D3 andhBMEC cell lines were transfected and infected as previously described(Coureuil et al., 2010, Cell, 143:1149-1160; Doulet et al., 2006, J CellBiol, 173-627-637). Plasmid encoding human CD147 was kindly provided byDr Bukrinsky and plasmid encoding β2-Adrenergic receptor fused to YFP(p2AR-YFP) was described previously (Angers et al., 2000, Proc Natl AcadSci USA, 97:3684-3689). To silence the expression of CD147, CD44 andICAM-1, pools of four siRNA duplexes (ON-TARGET plus SMARTpool siRNAfrom Dharmacon) were used. The siCONTROL siRNA (Dharmacon) were used ascontrol. Two days after transfection, efficiency of knockdown wasassessed by FACS analysis and cells were infected concurrently. Whenindicated hBMEC cells were transfected with siRNA targeting the 3′untranslated region of CD147, 3′-GCUGUCUGGUUGCGCCAUUUU-5′ (SEQ ID NO: 1)or control siRNA 3′-AUGUAUUGGCCUGUAUAG-5′ (SEQ ID NO: 39) (Eurogentech)and 40 h later, cells were again either mock transfected (−) ortransfected with CD147 coding region cDNA. 8 hours later, surfaceexpression of CD147 was quantified by FACS analysis and cells wereinfected concurrently with Nm 8013 clone 12, capsulated, Opa-, Opc-serogroup C clinical isolate expressing a class I SB pilin. Mutants ofthis strain ApilC1, ApilC2, ApilE, ApilV, ApilX, AcomP and wild type Nmexpressing GFP were derived and cultured as described previously (Nassifet al., 1994, Proc Natl Acad Sci USA, 91:3769-3773; Mikaty et al., 2009,PloS Pathog, 5:e1000314; Helaine et al., 2005, Mol Microbiol, 55:65-77;Mairey et al., 2006, J Exp Med, 203:1939-1950).

Adhesion Assays

Meningococcal adhesion on hCMEC/D3, hBMEC and BB19 was assayed underboth static and shear stress conditions, as previously described(Coureuil et al., 2010, Cell, 143:1149-1160; Mairey et al., 2006, J ExpMed, 203:1939-1950; Lambotin et al., 2005, J Cell Sci, 118:3805-3816).When indicated meningococci were pre-incubated with 5 μg/ml solublerecombinant recombinant human CD147-Fc and ICAM-1-Fc chimera (comprisingthe Ig domains of CD147 or of ICAM-1 fused to the Fc domain of humanIgG1 and produced as disulfide-linked homodimers) before interactionwith hBMECs with additional 2 μg/ml soluble proteins. Meningococcaladhesion was quantified following a 30 min infection in staticconditions. To address the inhibitory effect of antibodies, hBMECs weregrown on IBIDI chambers, pretreated for 1 h with 10 μg/ml antibodiestargeting ICAM-1 (11C81) or CD147 (MEM-M6/1 and MEM-M6/6) and submittedto laminar flow (0.04 dynes/cm²) under an inverted microscope.GFP-expressing bacteria were introduced in the chamber under flow. After20 min, the number of adherent bacteria was determined. For adhesionassays on immobilised proteins, recombinant human CD147-Fc and ICAM1-Fcchimera were immobilised on glass slides using a modification of thetechnique described in Steffen et al., 2008, Curr Biol, 18:926-931.Briefly, slides were coated with 0.1% Poly-L Lysine, washed with PBS,crosslinked with glutaraldehyde (0.5%), washed, incubated with anti-Fcin assay buffer (PBS with 3% BSA) for 2 h, washed in assay buffer, andincubated overnight at 4° C. with 2 μg/ml chimeric protein. Slides werewashed before infection with meningococcal suspension of OD₆₀₀ 0.05.After incubation with bacteria, slides were washed three times andfixed. Bacteria were labelled and visualized with a Leica DMI6000microscope using a 63× oil-immersion objective. The number of adherentbacteria per field was quantified for 30 fields using ImageJ software.

Expression, Purification and Immobilisation of MBP-Pilin RecombinantProteins

Fragments of PilE, PilV, PilX and Comp lacking the region coding foramino acid residues 1 to 28 of the full-length proteins fused to themaltose-binding protein (MBP) were produced in E. Coli, purified onamylose resin (New England Biolabs) and immobilised on Staphylococcusaureus (ATCC 25923) expressing specific receptors for the Fc domain ofIgG as described before (Coureuil et al., 2010, Cell, 143:1149-1160).Coated bacteria were incubated with 1 μg of recombinant human CD147-Fcchimera for 30 min on ice. After 3 washes, bacteria were lysed withLaemmli buffer and the quantity of co-precipitated CD147-Fc protein wasassessed by immunoblot analysis. Bound CD147-Fc was quantified usingImage J software. When indicated, hCMEC/D3 cells were pretreated with 10μg/ml of MBP-Pilins for 15 min prior to infection with meningococcalsuspension of OD₆₀₀ 0.05 for 30 min. After incubation with bacteria,cells were washed three times and the number of adherent bacteria wasdetermined as described above.

Confocal Immunofluorescence Microscopy

hCMEC/D3, BB19 or hBMEC were grown to confluence on permanox coverslipsor on transwell filters. After the indicated treatments and/orinfection, cells were fixed and labelled as previously described(Lambotin et al., 2005, J Cell Sci, 118:3805-3816; Hoffmann et al.,2001, J Cell Biol, 155:133-143). Image acquisition and analysis wereperformed with a DM16000 microscope (Leica, 63×). Series of opticalsections were obtained with a confocal spinning disk microscope (Leica,63×). 3D reconstructions were obtained using Imaris software andquantification histograms with ImageJ software (NIH). Quantitativeanalysis of protein recruitment under bacterial colonies was determinedas the proportion of colonies positive for the protein of interestindicated. At least 50 colonies were observed per coverslip. Eachexperiment was repeated at least 3 times in duplicate or triplicate.

Infection of Human Brain Tissues

Fresh human brain sections were obtained from frontal lobe specimens ofmacroscopically and histologically normal brain (confirmed by aneuropathologist) of individuals referred to the Department of ForensicMedicine for unexplained out-of-hospital sudden death (consent formsML1094, PFS 10-008, ClinicalTrials.gov NCT00320099 from TheInstitutional Review Boards of the Poincaré Hospital, Versailles-SaintQuentin University and the French “Agence de la Biomédecine”).

After freezing of the brain tissue with isopentane cooled in liquidnitrogen, the sections, 7 μm thick, containing leptomeninges, corticalribbon and the underlying white matter were immobilised on superfrost™plus microscope slides and store at −80° C. Defrosted sections wererehydrated in PBS for 5 minutes and incubated for 1 h with mediumcontaining 0.1% BSA prior to infection with suspensions of bacteria(2.10⁷ bacteria in 150 μl of medium containing 0.1% BSA) for 1 h at 37°C. Sections were then gently washed horizontally 5 times and fixed inPAF 4% for 10 min at RT. When indicated, sections were treated for 1 hwith 10 μg/ml of antibodies targeting CD147 or ICAM-1 and then washed 3times prior to infection by bacteria. Adherent meningococci weredetected by chromogenic immunohistochemistry and immunofluorescenceanalysis. To perform chromogenic immunohistochemistry, after tissuerehydratation, sections were incubated overnight with the indicatedprimary antibodies and revealed using horseradish peroxidase coupledsecondary donkey anti-mouse antibody and an alkaline phosphatase donkeyanti-rabbit. DAB (brown color) and NBT/BCIP (blue color) were used aschromogens. For immunofluorescence analysis, brain sections wereincubated with the primary antibodies indicated for 2 h in PBS/BSA 0.1%.Alexa-conjugated phalloidin and DAPI (0.5 mg/ml) were added toAlexa-conjugated secondary antibodies for 1 h. After additional washing,coverslips were mounted in glycergel (Dako). Entire samples were scannedusing Nanozoomer 2.0 (Hamamatsu) and were further analysed using opticalmicroscopy, epifluorescence (Zeiss Axiovert, 40×) and confocal (spinningdisk Leica, 63×) microscopy. The marker specificities weresystematically confirmed by examining sections in which primary antibodywas replaced with isotype control Ig at the same concentration, and byimmunostaining of non infected tissues from the same donor.Quantification analysis of the fluorescently labelled bacteria thatadhered on a 1 mm² surface area was performed using ImageJ software(NIH). Results are presented as a mean of fluorescence per 100 μ², fromtwo independent experiments. 3D reconstructions were performed ondeconvoluted confocal stacks using Imaris software.

AlphaScreen® Assay

The AlphaScreen® technology was used to assess the interaction betweenMBP-Pilin recombinant fusion proteins and CD147-Fc-His or ALCAM-1-Fc-Hisas a control. The binding reaction was performed using white 384-wellOptiplates (PerkinElmer, Whalham, Mass., USA) in 20 μl (total reactionvolume). The AlphaScreen® reagents (anti-MBP-coated Acceptor beads andNickel chelate-coated Donor beads) were obtained from PerkinElmer.CD147-Fc-His (or ALCAM-1-Fc-His) and Pilin-MBP were prepared in 20 mMTris, pH 7.4, 20 mM NaCl. Donor beads (20 μg/ml) were incubated withCD147-His (0, 100 or 500 nM) for 45 min at room temperature. In parallelAcceptor beads (20 μg/ml) were incubated with Pilin-MBP (0, 50 or 500nM) for 1 h at room temperature in the Alphascreen® reaction buffer (25mM Tris, pH 7.4 at 20 ° C., 20 mM NaCl, 0.1% BSA and 0.05% Tween 20).Then, 10 μl of each of the interacting partners were added to the plate,allowed for incubation for either 2 h or overnight in the dark and atroom temperature. To perform antibody competition assay, 5 μl ofMEM-M-6/6 antibody (at various concentrations) were added toCD147-Fc-His-Donor beads for 30 min at room temperature beforeincubation with the Pilin-MBP Acceptor beads. Light signal was detectedby using the EnVision® multilabel plate reader (PerkinElmer). Allmanipulations involving AlphaScreen® beads were performed under subduedlighting.

Results and Discussion

To identify candidate receptors responsible for the initial attachmentof Nm Type IV pili expressed by endothelial cells, it has been takenadvantage of the inducible expression of a functional receptor on BB19,a transformed human brain endothelial cell line that is poorlypermissive to Nm adhesion. Treatment of BB19 cells with phorbol ester12-O-Tetradecanoylphorbol-13-acetate (TPA) strongly potentiated adhesionof Nm 8013 clone 12, a capsulated piliated clinical isolate of serogroupC (FIG. 1). This effect was inhibited by treatment with thetranscription inhibitor actinomycin D (FIG. 1). Furthermore, only thewild type strain and the bacterial pilC2 deletion mutant that harbouradhesive pili, adhered to TPA treated cells, in sharp contrast to thenon piliated pilE and the non adhesive pilC1 mutants, which failed to doso (FIG. 2). These data highlighted the transcriptional regulation of aputative adhesion receptor for meningococcal type IV pili. Using adifferential and quantitative large-scale analysis of expression of 10478 genes (see Methods), 211 up-regulated and 194 down-regulated geneswere identified in TPA-treated BB19 cells compared to untreated cells.Of the up-regulated genes, six encoded membrane-associated proteins,including CD147, a well known marker of the blood brain barrier (BBB),highly enriched on brain endothelial cells to which Nm preferentiallyadhere. CD147 is a member of the immunoglobulin (Ig) superfamily,containing two glycosylated Ig-like domains, involved in variousphysiological and pathological processes. It has been confirmed that TPAtreatment of BB19 cells up-regulated the expression of CD147.Furthermore adhesion of Nm to TPA-treated cells induced a massiverecruitment of CD147 to sites of bacterial adhesion. CD147 thus appearedto be an attractive receptor candidate for type IV pilus-mediated Nmadhesion.

To investigate the role of CD147 in the adhesion of Nm to endothelialcells, two cell lines were used as models. hCMEC/D3 is a fullydifferentiated human brain endothelial cell line derived from braincapillaries, which recapitulates the major phenotypic features of theBBB, and hBMEC, a human endothelial cell line isolated from bone marrowcapillaries. As expected, CD147 was predominantly enriched at cell-celljunctions of a polarized monolayer of hCMEC/D3 cells and a significantfraction of CD147 was present at the luminal surface. In cells infectedby Nm, CD147 accumulated at sites of meningococcal adhesion andco-localised with the β2-adrenergic receptor. Importantly, CD147 wasrecruited independently of the β2-adrenergic receptor expression andassociated-coupled signalling events, as expected of a putative receptormediating the initial bacterial adhesion (FIG. 3).

To establish whether CD147 was required for meningococcal adhesion,hCMEC/D3 and hBMEC cells were transfected with CD147-specific smallinterfering RNAs (siRNA) that knocked down 60 to 70% of the surfaceexpression of CD147 relative to control siRNA. Bacterial adhesion tocells in which CD147 was knocked down was drastically reduced (FIGS. 4and 5). This effect was restored by the re-expression of exogenous CD147(FIG. 5). Importantly, CD147 depletion also affected bacterial adhesionunder flow conditions, in a quantitative assay of the initial adhesionevents (FIG. 6), indicating that CD147 is essential to promote theinitial attachment of meningococci to human endothelial cells. Incontrast, a similar depletion of CD44 or ICAM-1 expression, two adhesionmolecules recruited at sites of bacterial adhesion upon type-IV mediatedsignalling events, did not affect meningococcal adhesion to eitherhCMEC/D3 or hBMEC cells, therefore supporting the specific effect ofCD147 depletion on meningococcal adhesion to human endothelial cells.

To further confirm the role of CD147 in Nm adhesion to endothelialcells, a recombinant soluble form of the extracellular domain of CD147(CD147-Fc) was used to specifically compete with the membrane-boundreceptor. Addition of CD147-Fc reduced bacterial adhesion to hBMEC cellsby 50% compared to non-treated cells or cells treated with controlsoluble ICAM-1-Fc (FIG. 7). An independent approach relied on the use oftwo CD147-specific antibodies, MEM-M6/1 and MEMM6/6, binding to theN-terminal and the C-terminal Ig domain of CD147, respectively.Interestingly, when added to hBMECs before infection, MEM-M6/6 inhibitedby 75% the initial attachment of Nm under flow conditions compared tocells pre-treated with anti-ICAM-1 control antibody (11C81), whereasMEM-M6/1 only reduced initial attachment by 20% (FIG. 8). Importantly,MEM-M6/1 and MEM-M6/6 both labelled CD147 expressed on hCMEC/D3 cellssimilarly. However only MEM-M6/1 efficiently labelled CD147 moleculesaccumulated at sites of bacterial adhesion, indicating that MEM-M6/6 andmeningococci compete for the same binding motif on CD147. Overall, ourdata indicate that CD147 is a critical endothelial receptor for theprimary attachment of Nm to human endothelial cells.

Next, we examined whether Nm could directly adhere on purified CD147-Fcmolecules immobilised on glass support. Remarkably, the number ofmeningococci adhering to CD147-Fc was 4 to 5 fold higher than the numberof bacteria adhering to ICAM-Fc or to the control condition with noimmobilised protein (negative) (FIG. 9). Consistent with a role formeningococcal type IV pili in this direct interaction, wild type andpilC2 mutant strains adhere to immobilised CD147-Fc, in contrast to pilEand pilC1 mutants (FIG. 10). To identify the CD147 ‘ligand(s)’ amongpilus components, we assessed the interaction between soluble CD147-Fcmolecules and purified recombinant pilins produced as fusion proteinswith the maltose binding protein (MBP) and immobilised on staphylococci(as described in Coureuil et al., 2010, Cell, 143:1149-1160) (FIG. 11).Interestingly, purified PilE interacted with CD147-Fc molecules. Asimilar interaction was observed with the minor pilin PilV, while nosignificant interaction was detected with the minor pilins PilX or ComP.To further analyse the relative contribution of each pilin tomeningococcal adhesion to endothelial cells, infections of hCMEC/D3cells with wild-type Nm were performed in the presence of purifiedpilins, to assess their ability to compete for binding to the membranereceptor (FIG. 12). Remarkably, addition of PilE or PilV reducedbacterial adhesion by 60% and 50%, respectively, whereas addition ofPilX or ComP proteins had no effect (FIG. 12). Consistently, a pilVdeletion mutant, which remains piliated unlike a pilE mutant, abolishedbacterial adhesion to hCMEC/D3 cells (FIG. 13). Altogether, theseresults point to a key role for a selective interaction between PilE andPilV with CD147 in meningococcal initial adhesion to endothelial cells.

To assess the relevance of this finding in Nm infection in humans, theability of live pathogenic meningococci to adhere on fresh brainsections obtained from frontal specimens of normal human brains wasexplored. Remarkably, bacteria predominantly established tightassociation with brain vessels of the leptomeninges (withinVirchow-Robin spaces) and of cortical region. Importantly, thislocalisation, highly reminiscent of neuropathological observations inbacterial meningitis, relied on the expression of both pilins PilE andPilV. Indeed, pilE or pilV deletion mutants adhered poorly to brainsections from the same donor compared to wild type Nm (FIG. 14). Wildtype bacteria formed colonies on the luminal side of meningeal andcortical brain vessels which are lined with endothelial cells expressingCD147. In addition, bacterial colonies were found in association withCD147+ leptomeningeal cells, but not with glial nor neuronal cells thatdo not express CD147, demonstrating that bacterial adhesion to freshhuman brain tissues closely correlated with CD147 expression. Finally,pre-treatment of brain sections with MEM-M6/6 prior to infection by Nmdrastically reduced brain colonisation, whereas MEM-M6/1 and 11C81antibodies were ineffective (FIG. 15). These results, in line with ourdata using cultured endothelial cells, confirmed that the C-terminaldomain of CD147 is essential for in situ infection of cerebral bloodvessels.

Overall, this study demonstrates that CD147 is an essential receptor forthe initial adhesion of Nm to brain and peripheral human endothelialcells by interacting directly with the pilus components PilE and PilV.Importantly, it provides the first demonstration in situ of an electivetargeting of cerebral blood vessels by pathogenic meningococci, relyingon specific interactions between microbial ligands and a brain receptor.Because of its key position in the pathophysiological cascade of tissuecolonization by Nm, CD147 becomes a major pharmacological target toinhibit meningococcal blood-borne infection of the brain and ofperipheral capillaries. The lack of animal model of meningococcalinfection has hampered the understanding of meningococcal pathogenesis.N. meningitidis interacts only with cells of human origin and onlyadheres to human cells, thus preventing the study of the role ofmeningococcal cell interaction in meningococcal pathogenesis usinganimal models.

A model using severe combined immunodeficient (SCID) mouse grafted withhuman skin have been set up. Previous reports have shown that 4-6 weeksafter engraftment the human skin graft maintained characteristics ofnormal human skin, including its micro-vasculature (Yan et al., 1993, JClin Invest, 91: 986; Gilet et al., 2009, J Invest Dermatol, 129: 879.Briefly, normal human adult skin removed during plastic surgery wasgrafted on the back of SCID mice. One month later, mice are infected byintraperitoneal or intravenous injection of capsulated piliatedmeningococci. Histopathological examination revealed that N.meningitidis specifically targets draining vessels of the xenograft (butnot that of normal mice skin), adhere to the endothelium and formobliterating colonies within the lumen of vessels. FIG. 16 clearly showsthat in SCID mice grafted with human skin, piliated capsulated bacteriawhich are able to interact with cells are in much higher number in thehuman skin than non piliated capsulated bacteria unable to interact withhuman skin. This was confirmed by histological examination. Indeed nonpiliated bacteria were unable to adhere in vivo to the human endothelialcells of the graft. These data demonstrate that piliation is requiredfor bacterial interaction in vivo with human cells.

The high level of the bacteraemia is believed to be of the utmostimportance for meningococcal pathogenesis. The virulence factors knownto allow the establishment of this bacteraemia are the capsule, thelipooligosaccharide, the factor H binding proteins and the ironchelation systems. These virulence factors allow the bacteria to survivein the extracellular fluids. Using the SCID mice model, we tested thehypothesis that the ability of the bacteria to interact and to multiplywithin the blood vessels was also participating in the establishment ofthe bacteraemia. As shown FIG. 17, when piliated bacteria are injectedin grafted human skin mice, the bacteraemia remains much higher thanwhen similar bacteria are injected in non-grafted mice, thus suggestingthat the interaction of the bacteria with the human skin allows theestablishment of the bacteraemia. The role of the type IV pili wasconfirmed by the fact that injection of non piliated meningococci(unable to adhere to endothelial cells) are clearing the bacteria fromthe bloodstream unlike what is observed with piliated bacteria. Toconfirm the role of this interaction in meningococcal pathogenesis, thevirulence of piliated meningococci between injected grafted mice orinjected non-grafted mice has been compared. As shown FIG. 18, 2 out of3 grafted mice died whereas all non grafted mice survived.

Altogether these data demonstrate that the establishment of ameningococcal bacteraemia requires not only the capsule,lipooligosaccharide, factor H binding proteins and iron chelationsystem, but also pili which promote the interaction of the bacteria withthe endothelial cells. In addition, this interaction with endothelialcells is essential for meningococcal pathogenesis (FIG. 18). These datasuggest that inhibiting this interaction will prevent meningococcalinfection, and that a vaccine aiming at decreasing the meningococcalcell interaction will be efficient at preventing meningococcalinfection.

Finally, an Alphascreen test allowing to measure the interaction betweenpilins and CD147 ectodomain has been developped. A dose dependentinteraction between CD147-Fc and both PilE and PilV, and not with theother minor pilins PilX and Comp, was detected using Alphascreen®technology, while no interaction was detected between PilE or PilV witha control protein. As expected, purified MBP-PilE-SA and MBP-PilE-SBchimera similarly interacted with CD147-Fc. Furthermore, interactionbetween CD147 and PilE can be successfully inhibited by the addition ofthe MEM-M6/6 antibody directed against the Ig proximal domain of CD147.These results suggest that the Alphascreen test can be used for thehigh-throughput screening of inhibitors of the interaction between typeIV pilus-associated proteins and CD147.

What is claimed is:
 1. A method for preventing or treating ameningococcal bacteremia and/or infection in an individual in needthereof, comprising administering a therapeutically effective amount ofan immunogenic PilV polypeptide comprising SEQ ID NO: 32, or a fragmentthereof, or SEQ ID NO: 34, or a fragment thereof, capable of raising inthe individual an immune response with antibodies that prevent theinteraction between said type IV pilus-associated protein and the CD147receptor.
 2. The method according to claim 1, wherein the immunogenicPilV polypeptide comprises or consists of a) SEQ ID NO: 32, or b) afragment of at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,75 consecutives amino acids of the sequence SEQ ID NO: 32, or c) asequence that is at least 80, 85 or 90% identical to the sequence of (a)or (b).
 3. The method according to claim 1, wherein the immunogenic PilVpolypeptide comprises or consists of a) SEQ ID NO: 32, or b) a fragmentof at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75consecutives amino acids of the sequence SEQ ID NO: 32, or c) a sequencethat is at least, 95 or 99% identical to the sequence of (a) or (b). 4.The method according to claim 1, wherein the immunogenic PilV fragmentcomprises or consists of a) SEQ ID NO: 34, or b) a fragment of at least10, 15, 20, 25 consecutives amino acids of the sequence SEQ ID NO: 34,or c) a sequence that is at least 80, 85, 90% identical to the sequenceof (a) or (b).
 5. The method according to claim 1, wherein theimmunogenic PilV fragment comprises or consists of a) SEQ ID NO: 34, orb) a fragment of at least 10, 15, 20, 25 consecutives amino acids of thesequence SEQ ID NO: 34, or c) a sequence that is at least 95 or 99%identical to the sequence of (a) or (b).
 6. The method according toclaim 1, further comprising administering at least one anti-bacterialcompound, either sequentially or simultaneously.
 7. The method accordingto claim 6, wherein said anti-bacterial compound is selected from thegroup consisting of meningococcal PilE, PilV, PilX, PilC, ComP, fHbp,PorA, NHBA, NadA, MafA, NspA, HmbR, TbpB, and AusP, or an immunogenicfragment thereof.
 8. The method according to claim 6, wherein saidanti-bacterial compound comprises or consists of PilE or an immunogenicfragment thereof.
 9. The method according to claim 1, wherein saidimmunogenic PilV polypeptide is formulated in a pharmaceuticallyacceptable composition.